Sulfated-Oxysterol and Oxysterol Sulfation by Hydroxysterol Sulfotransferase Promote Lipid Homeostasis and Liver Tissue Regeneration

ABSTRACT

Methods and compositions for the prevention and treatment of liver damage or disease in a subject in need thereof are provided. The methods involve providing the sulfated oxysterol 25-hydroxycholesterol-3-sulfate (25HC3S) to the subject e.g. by 1) administering 25HC3S to the subject; or 2) overexpressing, in the subject, the hydroxysterol sulfotransferase enzyme SULT2B1b, which catalyzes the sulfation of 25-hydroxycholesterol (25HC) to form 25HC3S.

The present application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/472,293, filed Apr. 6, 2011, and U.S.Provisional Application No. 61/604,711, filed Feb. 29, 2012, thecomplete disclosures of both of which are expressly incorporated byreference herein in their entirety. The present application alsoexpressly incorporates by reference herein the entire disclosure of U.S.Application Nos. 20070275939, filed Apr. 24, 2007, and 20100273761,filed Feb. 19, 2010

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No. ROIHL078898 awarded by the National Institutes of Health to Shunlin Ren.Part of the work performed during the development of this inventionutilized U.S. government funds. The government may therefore havecertain rights in this invention.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to the prevention and treatment of liverdisease or damage. In particular, the invention provides methods ofproviding the sulfated oxysterol 25-hydroxycholesterol-3-sulfate(25HC3S) to a subject in order to prevent or treat liver disease ordamage such as nonalcoholic fatty liver disease (NAFLD), to facilitaterecovery after hepatectomy surgery and to promote lipid homeostasis.

Background of the Invention

The liver is a vital organ present in vertebrates and some otheranimals. This organ plays a major role in metabolism and has a number offunctions in the body, including glycogen storage, decomposition of redblood cells, plasma protein synthesis, hormone production, themaintenance of lipid homeostasis, detoxification, and production ofbiochemicals necessary for digestion. As a result of the wide-rangingand vital functions of this organ, subjects with liver disease or damagecan experience drastic and debilitating health consequences. Althoughliver dialysis can be used in the short term, there is currently no wayto compensate for the long term absence of liver function.

Damage to the liver can occur due to and/or be associated with a varietyof factors such as exposure to various toxins, excessive consumption ofalcohol, obesity, high fat diets, viral infections, hereditary factors,cancer, the long-term use of certain medications, trauma as the resultof an accident or combat, etc. One particular liver disease that iscurrently of major concern is nonalcoholic fatty liver diseases (NAFLD).NAFLD is characterized by the accumulation of lipids (e.g.triglycerides) in liver tissue. This syndrome is associated with obesityand is currently estimated to affect almost one-quarter of the generalUnited States population. The spectrum of NAFLD ranges from simplenonprogressive steatosis to progressive steatohepatitis (NASH) that ischaracterized by inflammation and results in liver cirrhosis andhepatocellular carcinoma. Lowering triglyceride levels andanti-inflammatory responses are important elements of successful NAFLDprevention and therapy. However, this option is unlikely to be adoptedby many individuals in the developed world. A large number of medicaltreatments for NAFLD have been studied and, while many appear to improvebiochemical markers such as alanine transaminase levels, most have notbeen shown to reverse histological abnormalities or reduce clinicalendpoints.

Currently, the only long-term option for treating severe liver damage isliver transplantation, which may involve receipt of an entire organ froma deceased donor, or receipt of a lobe or liver tissue donated by a livedonor. While transplantation can be successful, especially in view ofthe compensatory growth capabilities of liver tissue, the procedure isdrastic, requiring major surgery and subsequent monitoring and treatmentto avoid rejection.

There is obviously a need in the art to develop techniques to prevent ortreat liver damage such as that which results from NAFLD, or from othercauses, and to promote lipid homeostasis.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for use in theprevention and/or treatment of liver disease and damage in subjects inneed thereof. The methods involve increasing the level of thecholesterol metabolite, 25-hydroxycholesterol-3-sulfate (5HC3S) in thesubject. In some embodiments, 25HC3S is increased (elevated) by directadministration of the compound to the subject. In other embodiments,25HC3S is increased indirectly by overexpression, in the subject, of thehydroxysterol sulfotransferase enzyme SULT2B1b, which catalyzes thesulfation of the endogenous substrate 25-hydroxycholesterol (25HC) toform 25HC3S. This second embodiment may optionally also compriseadministering exogenous 25HC substrate to the subject.

As described above, the liver is responsible for the maintenance oflipid homeostasis in the body, and the active agents that areadministered as described herein appear to act principally on the liver.As such, the compounds may be used to both prevent and treat disease anddamage of the liver per se (e.g. NAFLD), and to prevent and treatdiseases associated with excessively high levels of circulating lipids,i.e. prevent or treat hyperlipidemia and associated disorders such asartherosclerosis.

In one or more aspects, the invention involves a method for promotingliver cell proliferation or liver tissue regeneration in a subject,comprising: elevating a level of 25-hydroxycholesterol-3-sulfate(25HC3S) in a subject in need of at least one of liver cellproliferation and liver tissue regeneration in order to promoteproliferation of liver cells or regeneration of liver tissue in saidsubject. In one embodiment, elevating a level of 25HC3S is performed byadministration of exogenous 25HC3S to said subject. In one embodiment,25HC3S is administered in an amount ranging from 0.1 mg/kg to 100 mg/kg,based on body mass of said subject. In another embodiment, 25HC3S isadministered in an amount ranging from 1 mg/kg to 10 mg/kg, based onbody mass of said subject. In other embodiments, administrationcomprises at least one of oral administration, enteric administration,sublingual administration, transdermal administration, intravenousadministration, peritoneal administration, parenteral administration,administration by injection, subcutaneous injection, and intramuscularinjection. In another embodiment, elevating a level of 25HC3S isperformed by providing hydroxycholesterol sulfotransferase 2B1b(SULT2B1b) and 25-hydroxycholesterol (25HC) to said subject. In yetother embodiments, elevating a level of 25HC3S is performed by promotingoverexpression of SULT2B1b in said subject and providing 25HC to saidsubject. In some embodiments, elevating is performed before, during orafter liver surgery in said subject. In one embodiment, the liversurgery comprises liver transplant surgery. In other embodiments, thesubject has at least one of cirrhosis, liver injury, and hepatitis. Inyet other embodiments, elevating is performed using a viral vectorcomprising a nucleic acid sequence coding for SULT2B1b.

The invention further involves a method for promoting liverregeneration, comprising: administering to a subject a nucleic acidsequence coding for SULT2B1b, wherein said administering is performedsuch that said nucleic acid sequence is selectively expressed in livercells in said subject. In one embodiment, administering is performedbefore, during or after liver surgery in said subject. In anotherembodiment, the liver surgery comprises liver transplant surgery. In yetother embodiments, the subject has at least one of cirrhosis, liverinjury, and hepatitis. In some embodiments of the invention,administering is performed using a viral vector.

The invention also involves a method for treating or preventinghyperlipidemia or fatty liver disease or malady resulting fromhyperlipidemia or fatty liver disease, comprising: providing a subjectwith a sufficient amount of a sulfotransferase to elevate endogenouslevels of 25HC3S in said subject, wherein said elevated endogenouslevels of 25HC3S are sufficient to decrease lipid synthesis in saidsubject so as to reduce serum and hepatic lipid levels in said subject,or to reduce one or more regulators of lipid metabolism in said subject.In one embodiment, the sulfotransferase is SULT2B1b. In someembodiments, providing is performed by administering said subject anucleic acid sequence coding for SULT2B1b, wherein said administering isperformed such that said nucleic acid sequence is expressed in livercells in said subject. In other embodiments, the method also comprisesadministering 25HC to said subject. In yet other embodiments,administering is performed using a viral vector. In some embodiments,the hyperlipidemia is selected from hypercholestolemia andhypertrigyceridemia. In some embodiments, the fatty liver disease isnon-alcoholic fatty liver disease. In other embodiments, the maladycomprises at least one malady selected from atherosclerosis, stroke,gall stones, diabetes, inflammatory bowel disease, and non-alcoholicsteatohepatitis. In some embodiments, administering is performed suchthat said nucleic acid sequence is selectively expressed in at least oneof liver tissue, lung tissue, and aorta tissue in said subject.

Other features and advantages of the present invention will be set forthin the description of invention that follows, and in part will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the description ofinvention that follows, in reference to the noted plurality ofnon-limiting drawings, wherein:

FIG. 1A-F. Effects of 25HC3S and 25HC on the serum lipoprotein profilein HFD-fed mice. Eight-week-old C57BL/6J female mice were fed a high fatdiet (HFD) for 10 weeks, treated with either 25HC3S, 25HC or vehicletwice and fasted over night, n=15-17. Serum lipoprotein profiles wereseparated by HPLC with a Superose 6 column (A and B), and each fractionwas collected for the measuring of concentration of triglyceride (TG)(Panels C and D) and cholesterol (CHOL) (E and F). The data representone of three separate experiments.

FIG. 2. HPLC Analysis of 25HC3S and 25HC levels in the treated miceliver tissues. Animals were treated as described in FIG. 1. Totalneutral lipids were extracted with chloroform/methanol mixture andanalyzed by HPLC. 24-hydroxycholesterol (24HC), 25-hydroxycholesterol(25HC), 27-hydroxycholesterol (27HC) and 7α-hydroxycholesterol (7α-HC)were used as standard controls; and testosterone in the chloroform phasewas used as an internal control. Oxysterols in the chloroform phase fromvehicle-treated, 25HC-treated, and 25HC3S-treated mouse liver weredetermined (Left panel). Chemical synthesis of 25HC3S was used asstandard control in water/methanol phase. 25HC3S in water/methanol phasefrom vehicle-treated, 25HC-treated and 25HC3S-treated mice liver weredetermined (Right panel). The data represent one of three separateexperiments.

FIG. 3A-D. The effects of 25HC3S and 25HC on gene expressions in lipidmetabolism in liver tissues. Animals were treated as described inFIG. 1. Specific protein levels in cytoplasmic and nuclear extracts weredetermined by Western blot analysis. A and C, protein levels of ACC1 andFAS normalized to β-actin; B and D, protein levels of SREBP1 and SREBP2normalized to Lamin B1. All the values are expressed as mean±SD. Thesymbol #represents p<0.05 versus chow diet-fed vehicle-treated miceliver; * p<0.05 versus HFD-fed vehicle-treated mice liver; (n=3).

FIG. 4A-F. The effects of long term-treatment with 25HC3S on mouse bodymass and food intake. C57BL/6J female mice were fed with HFD, separatedto two groups, and treated with either 25HC3S or vehicle once everythree days for 6 weeks. During these 6 weeks, the total food intake (A)and the body weight were monitored (B). After 5 hours fasting, the liverweight was determined (C), the plasma alkaline phosphatase (ALK) (D),alanine aminotransferase (ALT) (E) and aspartate aminotransferase (AST)(F) were determined. All the values are expressed as mean±SD.Statistical significant difference (p=0.017, n=16, by pair t-test). *p<0.05 and ** p<0.01 versus HFD-fed vehicle-treated mouse liver.

FIG. 5A-F. A long-term treatment with 25HC3S decreases lipidaccumulation in liver tissue in mouse NAFLD models. Animals were treatedas described in FIG. 4. Hepatic triglyceride (A); free fatty acid (B);total cholesterol (C); free cholesterol (D); and cholesterol ester (E)were measured. Each individual level was normalized by liver weight. Allthe values are expressed as mean±SD. # p<0.001 versus chow-fedvehicle-treated mice. * p<0.05 and ** p<0.01 versus HFD-fedvehicle-treated mice liver. In morphology studies (F), liver sectionsfrom chow diet fed (chow), high fat diet fed (HFD) and high fat diet fedwith 25HC3S treated (HFD-25HC3S) mice were stained by H&E staining.Arrows indicate unstained lipid inclusions.

FIG. 6A-D. Effect of adenovirus infection on liver toxicity. C57BL/6mice, 12 w, were infected with Adenovirus through tail vein injection.Each group contains 3 mice. FIGS. 6A and B show the liver-specificcytosolic enzyme activities of alkaline phosphatase (ALP), alaninetransaminase (ALT), and aspartate transaminase (AST) in serum of mice,and the ratio of liver weight to body weight 3, 6, 12, and 24 days afteradenovirus injection (1×10⁸ pfu). FIGS. 6C and D show the ALP, ALT, andAST activities in the serum and the ratio of liver to body weight 6 daysafter injection with different amount adenovirus (0, 1×10⁶, 1×10⁷, 1×0⁸,1×10⁹ pfu/mouse). * P<0.05, ** P<0.01 vs. 0 day or vehicle.

FIG. 7A-C. Determination of SULT2B1b expression in different tissuesafter infection with AdSULT2B1b. C57BL/6 mice, 12 w, were infected withAd-Control or Ad-Sult2B1b (1×0⁸ pfu) as indicated. (A)Immunohistochemistry analysis of SULT2B1b protein expression indifferent tissues 6 days after infection with Ad-Control or Ad-Sult2B1b.(B and C) SULT2B1b protein levels in different tissues were analyzed byWestern blot. The data represent one of three separate experiments. *P<0.05, ** P<0.01 vs. Ad-Control.

FIG. 8A-C. Effect of SULT2B1b overexpression on lipoprotein cholesteroland triglycerides in serum by HPLC. C57BL/6 mice and LDL^(−/−) mice, 8w, fed with high cholesterol diet (HCD) or high fat diet (HFD) for 10weeks, then the mice were infected with Ad-control or Ad-SULT2B1b (1×10⁸pfu) in the presence or absence of 25HC as indicated. Each groupcontains 5-6 mice. (A) HPLC analysis of the lipoprotein cholesterol(VLDL, LDL, and HDL) in serum both in C57BL/6 mice and LDL^(−/−) mice.(B) HPLC analysis of the lipoprotein tryglycerides (VLDL, LDL, and HDL)in serum both in C57BL/6 mice and LDL^(−/−) mice. (C) Protein assay inserum was used as internal control. The data represent one of threeseparate experiments.

FIG. 9A-D. Effect of SULT2B1b overexpression on lipid levels in livertissue. C57BL/6 mice and LDLR^(−/−) mice, 8 w, fed with high cholesteroldiet (HCD) or high fat diet (HFD) for 10 weeks, then the mice wereinfected with Ad-control or Ad-SULT2B1b (1×10⁸ pfu) in the presence orabsence of 25HC as indicated. Each group contains 5-6 mice. (A) H&Estaining analysis of total lipids in liver tissue. (B-D) Triglycerides(TG), free fatty acids (FFA), total cholesterol (TC) and freecholesterol (FC) in liver both in C57BL/6 mice and LDL^(−/−) mice wereanalyzed as described in Methods. * P<0.05, ** P<0.01 vs. Ad-Control.

FIG. 10A-D. Effect of SULT2B1b overexpression on oxysterol and sulfatedoxysterol levels in liver tissue. C57BL/6 mice and LDLR^(−/−) mice, 8 w,fed with high cholesterol diet (HCD) or high fat diet (HFD) for 10weeks, then the mice were infected with Ad-control or AdSULT2B1b (1×0⁸pfu) in the presence or absence of 25HC as indicated. Each groupcontains 5-6 mice. Total intracellular neutral lipids were extracted byadding 10 volumes of chloroform/methanol mixture (2:1, v/v). (A and B)Oxysterols in chloroform phase were analyzed by HPLC. (C and D) Sulfatedoxysterols in methanol/water phase were analyzed by HPLC. 7KC, 6βHC, and25HC were used as standard controls. The data represent one of threeseparate experiments.

FIGS. 11A and B. Effect of SULT2B1b overexpressoin on gene expressionsinvolved in lipid metabolism at protein level. C57BL16 mice and LDLR′mice, 8 w, fed with high cholesterol diet (HCD) or high fat diet (HFD)for 10 weeks, then the mice were infected with Ad-control or Ad-SULT2B1b(1×10⁸ pfu) in the presence or absence of 25HC as indicated. (A)Westernblot analysis of nuclear extracts and cytosolic proteins with specificantibodies against LXRa, ABCA1, SREBP-1, SREBP-2 and SULT2B1b. (B)Quantitative analysis for western blot data. * P<0.05, ** P<0.01 vs.Con.

FIGS. 12 A and B. A, Pharmacokinetics and B, tissue biodistribution ofradioactivity following intravenous injection of [³H]-25HC3S and 25HC3Sin mice. Each point represents one animal.

FIG. 13A-D. Expression levels of cell cycle-related genes in response to25HC3S in mouse liver tissues. Mice have been treated for 48 h with25HC3S at different concentrations as indicated. mRNA levels of FoxM1b(A), CDC25b (B), cyclin A (C), and c-myc (D) were analyzed by RTqPCR atthe end of the treatment. The results are shown as mean±S.D.(n=3-5/group) * P<0.05 vs. mRNA expression at 0 mg/kg concentration.

FIG. 14 A-C. Effect of exogenous 25HC3S on PCNA labeling index in mouseliver tissues. A, B and C show representative photomicrographs fromPCNA-stained liver sections of normal mouse group (A), vehicle (PBS 10%ethanol, 48 h) group (B), and 25HC3S (5 mg/kg, 48 h) group; (C) PCNAlabeling index obtained from liver sections of each group. The resultsare shown as mean±S.D. (n=3-5/group) * P<0.05 vs. Normal mouse group.

FIGS. 15 A and B. Effect of endogenous 25HC3S on PCNA labeling index inmouse liver tissues. A, Mice have been infected for 5 d with eitherAd-Control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC(25 mg/kg) as indicated. B, PCNA labeling index obtained from liversections of each group were analyzed at the end of the treatment. Theresults are shown as mean±S.D. (n=3-5/group) * P<0.05 vs. correspondingAd-Control group.

FIGS. 16 A and B. Effect of exogenous 25HC3S on LXR activity and itstarget gene expressions in mouse liver tissues. Mice have been treatedfor 48 h with vehicle or 25HC3S (5 mg/kg) as indicated. (A) LXRa,SREBP-1c, ABCA1, and PCNA proteins were detected by western blot at theend of the treatment. (B) Western blot data were quantitativelynormalized to β-actin. The results are shown as mean±S.D.(n=3-5/group) * P<0.05 vs. Vehicle.

FIGS. 17A and B. Effect of endogenous 25HC3S on LXR activity and itstarget gene expressions in mouse liver tissues. Mice have been infectedfor 5 d with either Ad-Control or Ad-SULT2B1b (1×10⁸ pfu) in thepresence or absence of 25HC (25 mg/kg) as indicated. (A) LXRa, SREBP-1c,ABCA1, and PCNA proteins were detected by western blot at the end of thetreatment. (B) Western blot data were quantitatively normalized to1-actin. The results are shown as mean±S.D. (n=3-5/group) * P<0.05 vs.Ad-Control.

FIGS. 18A and B. Effect of LXR activation on 25HC3S-inducedproliferation. Mice have been treated for 48 h with either vehicle or25HC3S (5 mg/kg) in the presence or absence of T0901317 (5 mg/kg) asindicated. (A) LXRa, SREBP-1c, ABCA1, and PCNA proteins were detected bywestern blot at the end of the treatment. (B) Western blot data werequantitatively normalized to β-actin. The results are shown as mean±S.D.(n=3-5/group) * P<0.05 vs. Vehicle.

FIGS. 19A and B. SULT2B1b mRNA and protein levels in mouse liver tissuesfollowing infection. Mice were infected with Ad-Control or Ad-SULT2B1b.After the day as indicated, mice were sacrificed and liver tissues werecollected. (A) Relative mRNA level of SULT2B1b expression was measuredby RT-PCR. An equivalent of total RNA for each sample was loaded, and 25cycles for each sample were used for PCR analysis. (B) SULT2B1b proteinlevel was determined via western blot analysis. The results are shown asmean±S.D. (n=3-5/group) *P<0.05 vs. day 0.

FIG. 20A-C. Effect of SULT2B1b on PCNA expression in mouse livertissues. At the indicated day following infection with Ad-Control orAd-SULT2B1b, mice were sacrificed and liver tissues were harvested. (A)Representative photomicrographs from PCNA-stained liver sections(20×optical field); (B) Percentage number of PCNA-positive cellsobtained from liver sections; (C) SULT2B1b and PCNA protein levels weredetermined via western blot analysis. The results are shown as mean±S.D.(n=3-5/group) *P<0.05 vs. day 0.

FIG. 21A-E. Effect of SULT2B1b overexpression on proliferative geneexpressions in mouse liver tissues. Following infection with Ad-Controlor Ad-SULT2B 1 b, mice were sacrificed at the days as indicated. TotalmRNAs were prepared from liver tissues, and each mRNA level was analyzedby RTqPCR. (A) PCNA; (B) FoxM1b; (C) CDC25b; (D) Cyclin A; (E) MMP-9expressions at mRNA level. The results are shown as mean±S.D.(n=3-5/group) *P<0.05 vs. day 0.

FIGS. 22A and B. Effect of SULT2B1b overexpression on LXR and its targetgene expressions in mouse liver tissues. Mice were sacrificed at thedays as indicated following Ad-Control or Ad-SULT2B1b infection. (A andB) The hepatic SULT2B1b, PCNA, LXRa, ABCA1 and SREBP1 protein levels inthe control (black bars) and SULT2B1b (gray bars) groups were analyzedby western blot. The results are shown as mean±S.D. (n=3-5/group)#P<0.05 vs. Con.

FIGS. 23A and B. Role of LXR signaling in SULT2B 1 b-inducedproliferation in mouse liver tissues. Following infection withAd-Control or Ad-SULT2B1b, mice were then given intraperitonealinjections of 25HC or T0901317 (25 mg/kg) for every two days, andsacrificed at day 5. (A and B) The hepatic protein levels of SULT2B1b,PCNA, LXR and its target genes ABCA1 and SREBP1 were analyzed by westernblot. The results are shown as mean±S.D. (n=3-5/group) *P<0.05 vs.corresponding vehicle, #P<0.05 vs. Con.

FIG. 24A-F. Effect of siRNA-SULT2B 1 b on proliferation in PRH. PRH at aconfluency of 100% were transfected with siRNA-SULT2B1b or siRNA-controlfor 24 and 48 hours as indicated. (A) RtqPCR analysis of SULT2B1b mRNAlevel after siRNA-SULT2B 1 b transfection; (B-D) RTqPCR analysis of cellcycle regulatory gene mRNA levels, including CDK2, FoxM1b, cyclin A andPCNA; (F) Relative hepatocyte viabilities were measure by cell survivalassay. The OD value of cell cultured in normal (N) was arbitrarilyassigned as 100%. Data are the mean±S.D. of three determinations.*P<0.05 vs. N, #P<0.05 vs. siRNA-Control.

FIG. 25 A-C. Effect of SULT2B1b overexpression on proliferation in PRH.PRH at a confluency of 100% were infected with Ad-Control or Ad-SULT2B1bfor 24 and 48 hours as indicated. (A) SULT2B1b protein level wasdetermined via western blot analysis. (B) Relative hepatocyteviabilities were measured by cell survival assay. The OD value of cellscultured in normal (N) was arbitrarily assigned as 100%; (C) RTqPCRanalysis of cell cycle regulatory gene mRNA levels, including CDK2,FoxM1b, cyclin A and PCNA. Data are the mean±S.D. of threedeterminations. *P<0.05 vs. N, #P<0.05 vs. Con.

FIG. 26A-D. Effect of SULT2B1b on proliferation via LXR signalingpathway in PRH. PRH at a confluency of 100% were infected withAd-Control or Ad-SULT2B1b for 24 and 48 hours as indicated. (A and B)Western analysis of SULT2B1b, PCNA, LXRa, ABCA1 and SREBP1 proteinlevels; (C and D) 24 hours after infection, cells were treated with 25HC(3 μM) or T0901317 (1.5 μM) for another 24 hours, the protein levels ofSULT2B 1 b, PCNA, LXR and its target genes ABCA1 and SREBP1 wereanalyzed by western blot. The data of western blot represent one ofthree separate experiments. Data are the mean±S.D. of threedeterminations. *P<0.05 vs. 0 hour, #P<0.05 vs. Con.

FIG. 27A-D. Effect of SULT2B1b overexpression on proliferating cellnuclear antigen (PCNA) in mouse liver Mice were infected withAd-SULT2B1b (1×0⁸ pfu) as indicated. Each group contained 3-5 mice. A:Relative mRNA level of SULT2B1b expression was measured by RTqPCR. B:Percentage number of PCNA-positive cells obtained from liver sections atthe indicated time-points. C: Immunohistochemistry analysis of PCNAexpression on liver sections (20×optical field) in mice with SULT2B1binfection. D: Western blot analysis of SULT2B1b and PCNA expression atprotein levels. * Represents P<0.05 vs. time-point 0.

FIG. 28A-F. Effect of SULT2B1b overexpression on gene expressionsinvolved in proliferation. Mice were infected with Ad-SULT2B1b orAd-control (1×08 pfu) as indicated. Each group contained 3-5 mice. A-F:RTqPCR analysis of PCNA, Cyclin A, FoxM1b, CDC25b, MMP-9 and C-myc,expressions at mRNA level. * Represents P<0.05 vs. time-point 0.

FIG. 29A-E. Effect of siRNA-SULT2B1b on proliferation in PRH. PRH werecultured and transfected with siRNA-SULT2B1b for 24-48 hrs as described.(A) RTqPCR quantified the expression levels of SULT2B1b aftersiRNA-SULT2B1b transfection. (C-E) RTqPCR analyzed the gene expressionsrelated to cell cycle, including CDK2, FoxM1b, CyclinA and PCNA. *P<0.05 vs. Control

FIG. 30A-E. Effect of SULT2B1b overexpression on LXR and its target geneexpressions in normal mouse liver. Mice were infected with Ad-Control orAd-SULT2B1b (1×10⁸ pfu) as indicated. Each group contained 3-5 mice.Total protein and mRNA in liver tissue were extracted. A: Western blotanalysis of LXR and its target gene expressions at protein level. B-E:RTqPCR analysis of ABCG1, ABCA1, ABCG5 and SREBP-1 expression at mRNAlevel. The protein data represent one of three separate experiments.Con, the mice infected with Ad-control virus. Sult, the mice infectedwith Ad-SULT2B1b. * P<0.05 versus time-point 0.

FIG. 31A-H. Effect of SULT2B1b on proliferation via LXR signalingpathway in PRH. PRH were cultured and infected with Ad-control orAd-SULT2B1b adenovirus (MOI=10) for 24-48 hrs. (A) Western blot analysisof LXR, VSREBP1, ABCA1, SULT2B1b and PCNA protein levels. (B-E) RTqPCRanalyzed the gene expressions related to cell cycle, including PCNA,FoxM1b, CDK2, and CyclinA. (F-H) RTqPCR quantified the expression levelsof SREBP1, ABCA1 and ABCG5. Samples were harvest 24 hrs after adenovirusinfection, the protein levels of SULT2B1b, PCNA, LXR and its target genewere analysed by western blot. The data of western blot represents oneof three separate experiments. Con, PRH infected with Ad-control virus.Suit, PRH infected with AdSULT2B1b.* P<0.05 vs. Control

FIG. 32A-F. Effect of SULT2B1b overexpression on liver proliferationafter PH. Mice were infected with Ad-Control or Ad-SULT2B1b (1×10⁸ pfu)as indicated for 5 days. During the five days, mice were subjected to PHand allowed to live for 0, 1, 3 and 5 days. Each group contains 3-5mice. A: Immunohistochemistry analysis of PCNA expression on liversections (20×optical field) from mice with virus infection. B:Percentage number of PCNA-positive cells obtained from liver sections atthe indicated time-points. C: Liver regeneration after PH was monitoredby the ratio of liver to body weight. D-F: Acute liver injury after PHwas evaluated by measuring serum AST, ALT and ALP levels. H: RTqPCRanalysis of PCNA, C-myc, FoxM1b, CDC25b, Cyclin A and MMP-9 expressionsat mRNA level. The data represent one of three separate experiments.Con, the mice infected with Ad-control virus. Suit, the mice infectedwith Ad-SULT2B1b. * P<0.05 vs. Con.

FIG. 33A-F. Effect of SULT2B1b overexpression on hepatocyteproliferative gene expression after PH. Mice were infected withAd-Control or Ad-SULT2B1b (1×10⁸ pfu) for 5 days. During the five days,mice were subjected to PH and allowed to live for 0, 1, 3 and 5 days, asdescribed previously. Total RNAs were purified from mouse liver. Eachgroup contains 3-5 mice. A-F: RTqPCR analysis of PCNA, Cyclin A, FoxM1b,CDC25b, C-myc and MMP-9 expressions at mRNA level. * Represents P<0.05vs. con.

DETAILED DESCRIPTION

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly dictates otherwise.

The present invention provides methods for preventing and/or treatingliver damage or disease, and compositions for use in the methods.According to the invention, the sulfated oxysterol 25HC3S is provided toa subject who is experiencing or who is likely to experience a livermalady. Two basic methodologies for providing 25HC3S are encompassed. Inone embodiment, the sulfated oxysterol compound 25HC3S is administeredto the subject. In a second embodiment, a nucleic acid encoding thehydroxysterol sulfotransferase enzyme SULT2B1b is administered to thesubject in a manner that results in overexpression of SULT2B1b in thesubject. Overexpressed SULT2B1b catalyzes sulfation of the naturallyoccurring endogenous substrate 25HC within the subject, converting it to25HC3S, thereby increasing the concentration of 25HC3S in the subject.Optionally, exogenous 25HC substrate may be administered to the subjectin conjunction with administration of the nucleic acid. The inventionmay also encompass a treatment method in which both 25HC3S and a nucleicacid encoding SULT2B1b (with or without 25HC) are administered.

The present invention also provides methods for preventing and/ortreating lipidemia and/or diseases or damage associated with or causedby lipidemia. Without being bound by theory, the efficacy of the activeagents described herein (e.g. 25HC3S, or agents which produce 25HC3S) relowering lipids in serum and elsewhere appears to be related to theability of the agents to influence liver function in a positive manner,augmenting the liver's ability to maintain proper lipid homeostasis.

By “25-hydroxycholesterol-3-sulfate (25HC3S)” we mean a compound of thestructure:

25HC3S is described in detail, for example, in published United Statespatent application US-1020-0273761 (Ren et al.), the complete contentsof which is herein incorporated by reference in entirety.

The beneficial effects exerted by administration of the active agentsdescribed herein may include an increase in liver tissue re-growth orregeneration, and/or in an increase in total numbers of liver cells,and/or an increase in activity of liver cells, the increase either 1)occurring at a faster rate than would occur in the absence of thetreatment; or 2) resulting in the production of more, or morephysiologically active, liver cells than would occur in the absence ofthe increased amounts of 25HC3S. Alternatively, and/or in addition, thebeneficial effect may be a decrease in lipid levels e.g. in serum, inliver cells, etc. of the subject Regardless of the parameter that ismeasured to detect the beneficial effect, the effect is typically anincrease/decrease of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100%, compared to a suitablecontrol, e.g. a subject to whom the active agents described herein havenot been administered. For example, the beneficial effect may be anincrease in total liver weight or an increase is liver function of fromabout 10 to about 90%, compared to a control; or a decrease in totalserum lipids of from about 10 to about 90%. In some embodiments, theincrease or decrease is in the range of at least about 25 to 55%, orabout 30 to 50%, or about 30 to about 45%.

In some embodiments, the invention encompasses a method of increasing alevel of 25HC3S in a subject by providing the subject with the compound25HC3S. In other embodiments, the invention encompasses a method ofincreasing a level of 25HC3S in a subject by providing the subject withthe 25HC3S precursor 25HC. In yet other embodiments, the inventionencompasses a method of increasing a level of 25HC3S in a subject byproviding the subject with a translatable (expressable) nucleic acidsequence which encodes a SULT2B1b protein. Without being bound bytheory, it is believed that administration of such a nucleic acidresults in expression of the SULT2B1b in cells of the subject (e.g.liver cells), which in turn results in sulfation of 25HC to form 25HC3Sin clinically relevant amounts, i.e. amounts of 25HC3S which have abeneficial effect on liver cells or tissue of the recipient. Optionally,and in addition, the SULT2B1b substrate 25HC may be administered to thesubject with or in conjunction with the nucleic acid, e.g. in order toaugment the amount of substrate available for sulfation by SULT2B1b.

The amino acid sequences of suitable SULT2B1b proteins and exemplarynucleic acids which encode them are readily accessible to those of skillin the art. For example, Homo sapiens SULT2B1b is available as GenBankNo. NM-017465. An exemplary nucleic acid sequence encoding SULT2B1b isdescribed, for example, in issued U.S. Pat. No. 7,820,805 (Thomae, etal.), the complete contents of which is hereby incorporated byreference. Further, those of skill in the art will recognize that theentire enzyme need not be translated. Rather, functional portionsthereof (e.g. sections or portions of the enzyme which retain theability to sulfate 25HC) may be utilized. In addition, chimeric proteinswhich include SULT2B1b or SULT2B1b activity may also be employed.

In some embodiments, what is administered is “naked” DNA encoding aSULT2B1b protein. However, in most embodiments, what is administered isa vector which comprises nucleic acid sequences which encode a SULTB1bprotein, or an active form of the protein. Suitable vectors for use inthe invention include but are not limited to various plasmids, cosmids,viral- and bacterial-based vectors, etc. Typically, the vector is aviral-based vector. A number of suitable viral based vectors are knownin the art and have been used to successfully transfect mammalian cells.Among those are adenovirus, adenovirus-associated virus (AAV),papovaviruses, vacciniavirus, the insect-infecting baculovirus, andlentivirus, etc. The nucleic acid sequence that is utilized is typicallyoperationally linked to at least one promoter sequence which drivesexpression of the enzyme. Addition sequences such as leader sequencesmay, enhancer sequences, etc. may also be included in such constructs.The constructs may selectively express SULT2B 1 b, e.g. in liver tissue,lung tissue, aorta tissue, etc. In some embodiments, selectiveexpression may be due to the use of promoters that are selective forexpression in particular types of cells or tissue. Techniques andguidelines for such gene therapy are described, for example, in “Presentand future of adeno associated virus based gene therapy approaches.”Recent Pat Endocr Metab Immune Drug Discov. 2012 January; 6(1), 47-66.“Gene Delivery System: A Developing Arena of Study for the New Era ofMedicine” Recent Pat DNA Gene Seq. 2010 Jan. 2 [Epub ahead of print].“Nanoparticles in Gene Therapy Principles, Prospects, and Challenges”.Prog Mol Biol Transl Sci. 2011; 104:509-62.

In the embodiment which involves administration of both a nucleic acidencoding SULT2B1b plus the substrate or precursor 25HC, these twoentities (which may both be referred to as “active agents” herein) areadministered “in conjunction with” one another, by which we mean thatthey may be administered, for example, in a single composition, or asseparate compositions but at the same time or nearly the same time (e.g.within minutes or hours or one another). Alternatively, the two may beadministered in conjunction with each other if they are administered inany coordinated manner, e.g. the nucleic may be administered first and,several hours or a few days later, the substrate may be administered; orthe substrate may be administered first in order to “prime” or “load”the subject's system in readiness for substrate catalysis of theexpressed enzyme several hours or days later, etc. The details andprecise timing or scheduling is generally determined by skilled medicalpersonnel on a case by case basis, with precedent being provided by dataobtained from clinical trials.

Liver disorders that may be treated by the methods and compositions ofthe invention include but are not limited to: hepatitis, inflammation ofthe liver, caused mainly by various viruses but also by some poisons(e.g. alcohol); autoimmunity (autoimmune hepatitis) or hereditaryconditions; non-alcoholic fatty liver disease, a spectrum in disease,associated with obesity and characterized by an abundance of fat in theliver, which may lead to hepatitis, i.e. steatohepatitis and/orcirrhosis; cirrhosis, i.e. the formation of fibrous scar tissue in theliver due to replacing dead liver cells (the death of liver cells can becaused, e.g. by viral hepatitis, alcoholism or contact with otherliver-toxic chemicals); haemochromatosis, a hereditary disease causingthe accumulation of iron in the body, eventually leading to liverdamage; cancer of the liver (e.g. primary hepatocellular carcinoma orcholangiocarcinoma and metastatic cancers, usually from other parts ofthe gastrointestinal tract); Wilson's disease, a hereditary diseasewhich causes the body to retain copper; primary sclerosing cholangitis,an inflammatory disease of the bile duct, likely autoimmune in nature;primary biliary cirrhosis, an autoimmune disease of small bile ducts;Budd-Chiari syndrome (obstruction of the hepatic vein); Gilbert'ssyndrome, a genetic disorder of bilirubin metabolism, found in about 5%of the population; glycogen storage disease type II; as well as variouspediatric liver diseases, e.g. including biliary atresia, alpha-1antitrypsin deficiency, alagille syndrome, and progressive familialintrahepatic cholestasis, etc. In addition, liver damage from trauma mayalso be treated, e.g. damage caused by accidents, gunshot wounds, etc.Further, liver damage caused by certain medications may be prevented ortreated, for example, drugs such as the antiarrhythmic agent amiodarone,various antiviral drugs (e.g. nucleoside analogues), aspirin (rarely aspart of Reye's syndrome in children), corticosteroids, methotrexate,tamoxifen, tetracycline, etc. are known to cause liver damage.

In one embodiment, the methods are performed before, during or afterliver surgery in a subject. For example, the liver surgery may be livertransplant surgery and the subject that is treated may be a donor or arecipient; or the liver surgery may be surgery that removes disease ordamaged liver tissue, or that removes cancerous tumors, etc.

In some embodiments, the disease or condition that is prevented ortreated is or is caused by hyperlipidemia. By “hyperlipidemia” we meanthe condition of abnormally elevated levels of any or all lipids and/orlipoproteins in the blood. Hyperlipidemia includes both primary andsecondary subtypes, with primary hyperlipidemia usually being due togenetic causes (such as a mutation in a receptor protein), and secondaryhyperlipidemia arising from other underlying causes such as diabetes.Lipids and lipid composites that may be elevated in a subject andlowered by the treatments described herein include but are not limitedto chylomicrons, very low-density lipoproteins, intermediate-densitylipoproteins, low-density lipoproteins (LDLs) and high-densitylipoproteins (HDLs). In particular, elevated cholesterol(hypercholesteremia) and triglycerides (hypertriglyceridemia) are knownto be risk factors for blood vessel and cardiovascular disease due totheir influence on atherosclerosis. Lipid elevation may also predisposea subject to other conditions such as acute pancreatitis. The methods ofthe invention thus may also be used in the treatment or prophylaxis ofconditions that are or are associated with elevated lipids, thatinclude, for example, but are not limited to hyperlipidemia,hypercholesterolemia, hypertriglyceridemia, fatty liver (hepaticsteatosis), and metabolic syndrome cardiovascular diseases, coronaryheart disease, atherosclerosis, acute pancreatitis, various metabolicdisorders, such as insulin resistance syndrome, diabetes, polycysticovary syndrome, fatty liver disease, cachexia, obesity, atherosclerosis,arteriosclerosis, stroke, gall stones, inflammatory bowel disease, andthe like. In addition, various conditions associated with hyperlipidemiainclude those described in issued U.S. Pat. No. 8,003,795 (Liu, et al)and U.S. Pat. No. 8,044,243 (Sharma, et al), the complete contents ofboth of which are herein incorporated be reference in entirety.

Methods of treatment include administering to a subject in need thereofa therapeutically effective amount of at least one compound, activeagent or composition described herein. The compounds and/or activeagents may include one or more of 25HC, 25HC3S and/or nucleic acidsencoding SULT2B1b or an enzymatically active form thereof. The nucleicacids may be housed in a vector as described herein. The compounds ofthe invention can be used in the treatment or prophylaxis of a diseasestate or malady characterized by liver disease or damage, or associatedwith elevated plasma and/oror hepatic cholesterol or triglycerides.Generally, prophylactic or prophylaxis relates to a reduction in thelikelihood of the patient developing a disorder such as cirrhosis,hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, fatty liver,or metabolic syndrome or proceeding to a diagnosis state for thedisorder. For example, the compounds of the invention can be usedprophylactically as a measure designed to preserve health and preventthe spread or maturation of disease in a patient. It is also appreciatedthat the various modes of treatment or prevention of a disease such asliver disease, hyperlipidemia, hypercholesterolemia,hypertriglyceridemia, fatty liver, metabolic syndrome, etc. can mean“substantial” treatment or prevention, which includes total but alsoless than total treatment or prevention, and in which some biologicallyor medically relevant result is achieved. Furthermore, treatment ortreating as well as alleviating can refer to therapeutic treatment andprophylactic or preventative measures in which the object is to prevent,slow down (lessen) a disease state, condition or malady. For example, asubject can be successfully treated for hypercholesterolemia if, afterreceiving through administration an effective or therapeutic amount ofone or more active agents described herein, the subject shows observableand/or measurable reduction in or absence of one or more signs andsymptoms of the particular disease such as, but not limited to, improvedliver function, increased weight of liver tissue, increased number ofliver cells, reduced plasma total cholesterol, reduced plasmaLDL-cholesterol, increased hepatic expression of LDL receptor (LDLR),reduced plasma triglycerides, reduced morbidity and mortality, orimprovement in quality of life issues.

The methods of the invention may also include a step of identifying asubject that is in need of the treatments described herein, e.g. asubject who has symptoms of or is at risk of developing one of thediseases or conditions described herein. Such a subject may beidentified using any of the many testing or diagnostic methods that areknown, for example, by measuring/detecting raised triglycerides (e.g.triglyceride levels >150 mg/dL [1.7 mmol/L]), or by measuring/detectingreduced HDL cholesterol (e.g. <40 mg/dL [1.03 mmol/L] in males, and <50mg/dL [1.29 mmol/L] in females), by measuring/detecting raised bloodpressure (e.g. systolic BP>130 or diastolic BP>85 mm Hg), or bymeasuring/detecting raised fasting plasma glucose (FPG) (e.g. FPG>100mg/dL [5.6 mmol/L]), or by measuring/detecting body mass index (BMI)(e.g. a BMI>30 kg/m²); or by measuring liver function e.g. bydetermining various enzymes, etc. that are indicators of liver function,examples of which include but are not limited to test for albumin,alanine transaminase, aspartate transaminase, alkaline phosphatase,bilirubin, 5′ nucleotidase (5′NTD)5′, lactate dehydrogenase, tests forcoagulation and serum glucose, etc. A subject who is positive for one ormore of these indicators may be a candidate for receipt of thetreatments described herein. Those of skill in the art will recognizethat a medical professional will typically make a diagnosis based on aconstellation or collection of symptoms that are present in anindividual patient.

The present invention provides compositions for use in promoting liverhealing and regeneration, in lowering lipid levels, and/or insuppressing inflammatory responses e.g. lipid levels in blood or serum.In one embodiment, the compositions include substantially purified25HC3S as described herein, and a pharmacologically suitable(physiologically compatible) carrier. In another embodiment, thecompositions include substantially purified vector containing nucleicacid sequences that encode a SULT2B1b protein, or active form thereofand a pharmacologically suitable (physiologically compatible) carrier.In one embodiment, the compositions include substantially purified 25HCas described herein, and a pharmacologically suitable (physiologicallycompatible) carrier. The preparation of compositions suitable foradministration for both embodiments is well known to those of skill inthe art. Typically, such compositions are prepared either as liquidsolutions or suspensions, however solid forms such as tablets, pills,powders and the like are also contemplated. Solid forms suitable forsolution in, or suspension in, liquids prior to administration may alsobe prepared. The preparation may also be emulsified. The activeingredients may be mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredients. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanoland the like, or combinations thereof. In addition, the composition maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, and the like. If it is desiredto administer an oral form of the composition, various thickeners,flavorings, diluents, emulsifiers, dispersing aids or binders and thelike may be added. The composition of the present invention may containany such additional ingredients so as to provide the composition in aform suitable for administration. The final amount of active agent inthe formulations may vary. However, in general, the amount in theformulations will be from about 1-99%.

The compounds of the present invention may be obtained from varioussources. For example, 25HC is readily commercially available, e.g. fromResearch Plus, Inc. (Baynone, N.J.) or may be synthesized as described,for example by Ogawa (Steroids: 74 (2009) 81-87. 25HC3S may besynthesized, for example, as described herein (see Example 1), or asdescribed in published United States patent applications 20070275939 and20100273761 (Ren), the complete contents of both of which are herebyincorporated by reference in entirety.

The 25HC3S and/or 25HC compositions (preparations) of the presentinvention may be administered by any of the many suitable means whichare well known to those of skill in the art, including but not limitedto by injection (e.g. either systemically or via targeted injection intoor into the vicinity of the liver), intravenously, inhalation, orally,intravaginally, intranasally, by ingestion of a food product containingthe active agent, topically, by direct application to liver tissue afterresection but before closing the surgical wound, etc. In preferredembodiments, the mode of administration is by injection orintravenously. In addition, the compositions may be administered inconjunction with other treatment modalities such as substances thatboost the immune system, various chemotherapeutic agents, antibioticagents, growth factors, and the like. The amount of 25HC3S to beadministered may vary depending on characteristics of the subject towhom it is administered (for example, the species, gender, age, geneticmakeup, general health, etc.), as well as the disease or condition thatis being treated. However, the amount will generally be in the range offrom about 0.1 mg/kg to about 100 mg/kg, based on body mass of thesubject. In some embodiments, the amount ranges from about 1 mg/kg toabout 10 mg/kg, based on body mass of the subject.

Similarly, the compositions which comprise a nucleic acid bearing vectormay be administered by any of the many suitable means which are wellknown to those of skill in the art, including but not limited to byinjection, orally, intranasally, transcutaneously, intravenously,intraperitoneally, subcutaneously, intramuscularly, by inhalation, etc.In addition, these compositions may also be administered alone or incombination with other medicaments as described above for 25HC3Scompositions. If the vector is a viral vector, the dosage employed maygenerally be about 10³ to 10¹¹ viable organisms, preferably about 10³ to10⁹ viable virus particles (or pfu), as described (Shata et al., Vaccine20:623-629 (2001); Shata and Hone, J. Virol. 75:9665-9670 (2001)).

Subjects to whom the compositions of the invention are administered aregenerally mammals. In some embodiments, the mammal is a human. In otherembodiments, the subject is a non-human mammal, e.g. a companion pet, orother non-human animal that could benefit from the therapy.

The present invention will be further illustrated by way of thefollowing Examples. These examples are non-limiting and do not restrictthe scope of the invention. Unless stated otherwise, all percentages,parts, etc. presented in the examples are by weight.

Examples

The Examples provided below described the relationships among 25HC,25HC3S SULT2B1b and other moieties that are active in the liver. TheExamples also provide experimental evidence of the in vivo efficacy ofthe methods, as follows: I) the reduction/reversal of diet-induced serumand hepatic lipid accumulation in a mouse model of NAFLD is described inExamples 1 and 2. In Example 1, the reduction results from theadministration of 25HC3S; in Example 2, the reduction results from theoverexpression of SULT2B1b. II) the promotion of hepatic proliferationin a mouse model is described in Examples 3 and 4. In Example 3,proliferation results from the administration of 25HC3S; in Example 4,proliferation results from the overexpression of SULT2B1b. Example 5describes the promotion of liver regeneration after partial hepatectomyas a result of overexpression of SULT2B1b.

The following abbreviations are used in the Examples: 25HC,25-hydroxycholesterol; 27HC, 27-hydroxycholesterol; 25HC3S,5-cholesten-313, 25-diol 3-sulfate; ABCA(G): ATP-binding cassette,sub-family A(G); ACC1: acetyl-CoA carboxylase 1; ACOX1: acyl-CoA oxidase1; Ad-Control, adenovirus encoding 13-Gal; Ad-SULT2B 1 b, adenovirusencoding SULT2B 1 b; AP, alkaline phosphatase; ALT, alaninetransaminase; AST, aspartate transaminase; CDC25, cell division cycle25; CDC25b, cell division cycle 25b; CDKs, cyclin-dependent kinases;cDNA, Complementary DNA; CPT1: carnitine palmitoyltransferase 1; CYP7A1,cholesterol-7α-hydroxylase; CYP27A1: mitochondrial cholesterol27-hydroxylase; FABPI: fatty acid binding protein; FAS: fatty acidsynthase; FATP: fatty acid transport protein; FoxM1b, Forkhead Box m1b;G6Pase: glucose-6-phosphatase; GCK: glucokinase; GPAM:glycerol-3-phosphate acyltransferase HCD: high cholesterol diet; HFD:high fat diet; HMGR: 3-hydroxy-3-methylglutaryl-coenzyme a reductase; %IC/g, percentage of injected counts per gram of tissue; IL: interleukin;IκBα: nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor, alpha; i.p., intraperitonealy; i.v., intravenously; LDLR:low-density lipoprotein receptor; LXR, liver X receptor; MCAD: acyl-CoAdehydrogenase; MMP, matrix metalloproteinase; MOI, multiple ofinfection; mRNA, messenger RNA; MTTP: microsomal triglyceride transferprotein; NAFLD: nonalcoholic fatty liver disease; NASH: nonaclcoholicsteatohepatitis; NFκB: nuclear factor of kappa light polypeptide geneenhancer in B-cells; PCK1: phosphoenolpyruvate carboxykinase 1; PCNA,proliferating cell nuclear antigen; Pk1r: pyruvate kinase; PLTP:phospholipid transfer protein; PPAR: peroxisome proliferator-activatedreceptor; PRH, primary rat hepatocytes; RT-PCR, Reverse-transcriptionpolymerase chain reaction; RTqPCR, quantitative real-time PCR; SCD:stearoyl-CoA desaturase; SRB1: scavenger receptor class B, member 1;SREBP, sterol regulatory element binding protein; StarD1: steroidogenicacute regulatory protein, DI; SULT2B1b: sulfotransferase family,cytosolic, 2B, member 1b; siRNA, small interference RNA; TNFα tumornecrosis factor, alpha.

Example 1. Reversal of Diet-Induced Serum and Hepatic Lipid Accumulationby 5-cholesten-3β,25-diol 3-sulfate in Mouse Models of NonalcoholicFatty Liver Disease (NAFLD)

In mammals, sterol regulatory element-binding protein-1c (SREBP-1c)preferentially controls lipogenic gene expression; and regulates fattyacid and triglyceride homeostasis. Its role in fatty acid biosynthesisand the development of fatty liver disease is well documented.

Oxysterols act at multiple points in cholesterol homeostasis and lipidmetabolism. The oxysterol receptor, LXR, is sterol regulatedtranscription factor of lipid metabolism. Activation of LXR stimulatesthe expression of cholesterol efflux and clearance through ABCA1 andABCG5/8, but it also up-regulates the expression of SREBP-1c, which inturn regulates at least 32 genes involved in lipid biosynthesis andtransport. Therefore, activation of LXR by synthetic ligands couldreduce serum cholesterol levels to protect against atherosclerosis, butit also leads to hepatic steatosis and hypertriglyceridemia due to theinduction of fatty acid and triglyceride synthesis through activation ofSREBP-1c. Hepatocytes have a limited capacity to store fatty acids inthe form of triglycerides. Once the capacity is exceeded, cell damageoccurs. Excess amounts of intracellular free fatty acids trigger theproduction of reactive oxygen species (ROS), causing lipotoxicity andactivation of inflammatory signaling pathways, which ultimately lead toapoptosis. Thus, a compound that specifically inhibits the SREBP-1cpathway without activating LXR should be a good target for NAFLDtherapy.

The oxysterol, 5-cholesten-3(3,25-diol 3-sulfate (25HC3S), whichaccumulates in hepatocyte nuclei following overexpression of themitochondrial cholesterol delivery protein, StarD1, has been identified.This oxysterol is synthesized by sterol sulfotransferase SULT2B1b from25-hydroxycholesrol (25HC) by oxysterol sulfation. Overexpression ofSULT2B1b inactivates the response of LXRa to 25HC, and inhibits LXRtarget gene expressions, including SREBP-1c and ABCA1. However,over-expression of SULT2B1b or addition of exogenous 25HC3S decreasesboth SREBP-1 and SREBP-2 expression; blocks the SREBP-1c processing;represses the expression of key enzymes, including acetyl-CoAcarboxylase-1 (ACC-1), fatty acid synthase (FAS) and3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), involved in lipidmetabolism, subsequently decreasing neutral lipid and cholesterollevels. 25HC3S thus may act as a LXR antagonist and as a cholesterolsatiety signal; suppressing fatty acid and triglyceride syntheticpathway via inhibition of LXR/SREBP signaling. Moreover, 25HC3Sincreases IκBα expression; blocks TNFα-induced IκBα degradation; anddecreases nuclear NFκB levels. In contrast, 25HC acts in an oppositemanner: inducing IκBα. degradation and nuclear NFκB accumulations. Theseresults indicate that oxysterol sulfation is also involved ininflammatory responses and may represent a link between inflammatorypathways and the regulation of lipid homeostasis.

In the present study, we show that acute treatment with 25HC3Ssubstantially decreases serum triglyceride and cholesterol levels, andlong term treatment decreases lipid levels in liver tissues viaLXR-SREBP-1c signaling pathway in mouse NAFLD models. These findingsprovide strong evidence that the oxysterol sulfation product, 25HC3S, isa potent regulator involved in lipid metabolism.

Materials and Methods Chemical Synthesis of 5-cholesten-3/3, 25-diol3-sulfate

A mixture of 25-hydroxycholesterol (6.5 mg, 0.016 mmol) was dissolved indry pyridine (300 μl) and triethylamine-sulfur trioxide (3.5 mg, 0.019mmol) was stirred at room temperature for 2 hours. The solvents wereevaporated at 40° C. under nitrogen stream, and to the resulting syrupwas added 100 ml of alkalined CH₃OH, pH 8.0. After the pellets weredissolved completely and filtered, the products were purified by HPLCusing a C18 column with a gradient elution system. A binary system ofsolvent A (20% CH₃CN in H₂O, v/v) and solvent B (20% CH₃CN in CH₃OH,v/v) was used, beginning at 50% A and 50% B with an initial flow rate of1 ml/min for 10 min, increasing to 100% B and increasing the flow ratelinearly to 2 ml/min over a 30 min period, and followed by an additionalisocratic period of 20 min. The total duration was 60 min. 25HC3S wasobtained as its sodium salt (4.7 mg, 57%) and a white powder, and thestructure characterized by MS and nuclear magnetic resonance (NMR)spectroscopy analysis (not shown).

Animal Studies

Animal studies were approved by Institutional Animal Care and UseCommittee of McGuire Veterans Affairs Medical Center and were conductedin accordance with the Declaration of Helsinki, the Guide for the Careand Use of Laboratory Animals, and all applicable regulations. Toexamine the effect of 25HC3S on diet-induced lipid accumulation in seraand liver, 8-week-old female C57BL/6J mice (Charles River, Wilmington,Mass.) were randomly assigned to three groups: the first control groupwas fed a chow diet; the high fat diet (HFD) group was fed a HFD (HarlanTeklad, Madison, Wis.) containing 42% kcal from fat, 43% kcal fromcarbohydrate, 15% kcal from protein and 0.2% cholesterol; and the highcholesterol diet (HCD) group was fed a 2% cholesterol diet with 18% kcalfrom fat, 58% kcal from carbohydrate and 24% kcal from protein for 10weeks, respectively. All mice were housed under identical conditions inan aseptic facility and given free access to water and food. At the endof each period, the mice were intraperitoneally injected with vehiclesolution (ethanol/PBS; Vehicle), 25HC (25 mg/kg), or 25HC3S (25 mg/kg)for 2 times and fasted over night for acute treatment (n=15-17 for eachgroup) or once every three days for 6 weeks (n=16 for each group) andfasted 5 hrs for long-term treatment; and blood samples were collected.Serum triglyceride, total cholesterol, high densitylipoprotein-cholesterol, glucose, alkaline phosphatase (ALK), alanineaminotransferase (ALT), and aspartate aminotransferase (AST) weremeasured using standard enzymatic techniques in the clinical laboratoryat McGuire Veterans Affairs Medical Center. Lipoprotein profiles in serawere analyzed by HPLC as following described.

HPLC Analysis of Serum Lipoprotein Profiles

Mouse serum (100 μl) was injected on a Pharmacia Superose 6 HR 10/30FPLC column and eluted with 0.2 ml/min 154 mM NaCl pH 8.0, 0.1 mM EDTA,wavelength 280 nm. Fractions were collected for 1.2 min each. Thecholesterol assay was performed using 180 μl of each fraction plus 20 μlof a 10× solution of Wako Total Cholesterol E Reagent (Wako ChemicalsUSA, Richmond, Va.) in a 96 well plate, incubated at 37° C. and read at595 nm. The triglyceride assay was performed using 2 μl of each fractionplus 200 μl of Infinity™ Triglycerides Reagent (Fisher Scientific,Pittsburgh, Pa.) in a 96 well plate, incubated at 37° C. for 5 minutesand read at 500 nm.

Histomorphology Analysis

For each mouse, three specimens from different regions of the liver werecollected and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer atroom temperature overnight. The regions of the specimens werestandardized for all mice. The paraffin-embedded tissue sections (4 μm)were stained with hematoxylin and eosin. Quantification of hepaticlipids Liver tissues were homogenized, and lipids were extracted with amixture of chloroform and methanol (2:1), and filtered. The extracts,0.2 ml, were evaporated to dryness and dissolved in 100 l of isopropanolcontaining 10% of triton X-100 for cholesterol assay (Wako ChemicalsUSA, Richmond, Va.), the NEFA solution (0.5 g of EDTA-Na2, 2 g of TritonX-100, 0.76 ml of IN NaOH, and 0.5 g of sodium azide/l, pH 6.5) for freefatty acid assay (Wako Chemicals USA, Richmond, Va.), or isopropanolonly for triglyceride assay (Fisher Scientific, Pittsburgh, Pa.). All ofthe assays were performed according to the manufacturer's instructions,respectively. Each lipid concentration was normalized to liver weight.

Western Blot Analysis of Special Protein in Cytoplasmic and NuclearExtraction

Liver tissues were homogenized, and cytoplasmic and nuclear fractionswere extracted with NE-PER Nuclear and Cytoplasmic Extraction Kit(Fisher Scientific, Pittsburgh, Pa.). The expression levels of ACCI,FAS, SREBP-1 and SREBP-2 were detected with specific antibodies, whereβ-actin was used as loading controls for cytoplasmic fractions, andLamin B1 was used as loading controls for nuclear fractions. Eachpositive band was quantified by Advanced Image Data Analyzer (Aida,Straubenhardt, Germany).

Quantitative Real-Time Polymerase Chain Reaction (q-RT-PCR) Analysis

Total RNA was isolated with SV Total RNA Isolation Kit (Promega,Madison, Wis.), which included DNase treatment. Total RNA, 2 μg, wasused for the first-strand cDNA synthesis as recommended by themanufacturer (Invitrogen, Carlsbad, Calif.). Real-time RT-PCR wasperformed using SYBR Green as indicator on ABI 7500 Fast Real-Time PCRSystem (Applied Biosystems, Foster City, Calif.). All primer/probe setsfor real-time PCR were TaqMan gene expression assays (AppliedBiosystems, Foster City, Calif.). Amplifications of β-actin and GAPDHwere used as internal controls. Relative messenger RNA (mRNA) expressionwas quantified with the comparative cycle threshold (Ct) method and wasexpressed as 2^(−ΔΔCt). Suitable primers were utilized.

Analysis of hepatic oxysterols and oxysterol sulfates by HPLC

Mouse liver samples (400 mg) were digested by 2 mg/ml of proteinase K inPBS (1 ml) at 50° C. for 12 hours. To the digests, 40 ml ofchloroform:methanol=2:1 (v/v) was added and sonicated for 30 min. Afterbeing filtered the insoluble matter, 6 ml of water and 100 μl of 1M-K2CO₃ were added, shaken, and allowed to stand for about 3 hours forphase separation.

The water/methanol phase, which mainly contains sulfated oxysterols, wasevaporated under N₂. The residues were re-suspended in 20% methanol bysonication and passed through a Sep-Pak tC 18 cartridge (Waters,Milford, Mass.). After the cartridge was washed with 20% methanol, thesulfated oxysterol fractions were eluted with 60% methanol and taken todryness under N₂ stream below 40° C. The extracts were then solvolyzedin a mixture of acetone (1 ml), methanol (9 ml), and conc. HCl (20 μl)at 39° C. for overnight. After neutralized by 5% KOH in methanol, 30 μlof testosterone in chloroform solution (50 μg/ml) was added, andevaporated to dryness. The residues were re-suspended in 8 ml of hexane.The mixture was loaded onto a Waters Sep-Pak silica cartridge (400 mg)that had been washed with 2% isopropanol in hexane. The purifiedoxysterols fractions were eluted with 8 ml of isopropanol:hexane (1:9,v/v) and evaporated under N₂.

The chloroform phase, which mainly contains non-sulfated oxysterols, wasadded 30 μl of testosterone in chloroform solution (50 μg/ml) andevaporated under N₂ below 40° C. The residue was re-suspended in 8 ml ofhexane and passed through a Waters Sep-Pak silica cartridge to purifythe oxysterol fraction as described above.

The oxysterol samples thus obtained from each phase, methanol/waterphase and chloroform phase were derivatized to the corresponding3-Keto-Δ⁴ form with cholesterol oxidase essentially according to thereported method (27), and were analyzed by Water Alliance series 2695HPLC module fitted with 2487 Dual X absorbance detector (Waters,Milford, Mass.). The separation was carried out on an Ultraspere silicacolumn (5 μm, 4.6 mm id×250 mm; Beckman, Urbana, Ill.) andhexane:isopropanol:acetic acid=965:25:10 (by volume) as an eluent at aflow rate of 1.3 ml/min. The column temperature was kept constant at 30°C. and the enones were monitored at the absorption at 240 nm.

Statistical Analysis

All results were expressed as mean±standard deviation (SD). Western blotresults were repeated at least three times. Statistical analysis wasperformed with the Student t test. The p<0.05 values were consideredstatistically significant.

Results 25HC3S Administration Reduces Serum Lipid Levels in Mice Fed aHFD and HCD.

To investigate the effects of 25HC3S on hyperlipidemia and hepaticsteatosis in vivo, 8-week-old C57BL/6J female mice were fed a HFD toestablish a NAFLD model. After 10 weeks of feeding, we treated thesemice with 25HC3S, 25HC or vehicle twice in 14 hrs as acute treatment,and fasted the animals overnight. Caloric intake and weight gain weresimilar in all treated groups. As expected, in HFD group, compared tothe vehicle treatment, the acute treatment with 25HC3S significantlydecreased plasma triglyceride and cholesterol levels induced by HFD(Table 1). Plasma triglyceride levels were reduced compared to thoseseen in healthy chow diet-fed mice (Table 1). In contrast, 25HCsignificantly increased plasma cholesterol levels and did not changeplasma triglyceride levels (Table 1). The fasting serum glucose levelwas compared with those in chow-fed mice (Table 1). Interestingly, 25HCsignificantly decreased fasting glucose levels which were elevated byHFD, whereas 25HC3S had no effect. These results were further confirmedin the HCD group where plasma triglyceride levels were significantlydecreased by the acute treatment with 25HC3S (Table 1).

Liver function analysis showed that HFD raised serum ALT and AST levels.25HC treatment significantly increased the levels further (Table 1). Incontrast, there was no significant difference between 25HC3S-treated andvehicle-treated mice (Table 1). These results indicate 25HC inducesliver inflammation, leading a rise of liver injury, whereas 25HC3S doesnot.

TABLE 1 Serum Parameters of Mice Fed a HFD or HCD with or without 25HCor 25HC3S Triglycerides Cholesterol HDL-C Glucose ALK ALT AST (mg/dL)(mg/dL) (mg/dL) (mg/dL) (IU/L) (IU/L) (IU/L) Chow diet 39 ± 5   57 ± 5  51 ± 5   196 ± 32 93 ± 15 32 ± 5 175 ± 11 HFD 53 ± 11***  106 ± 16*** 90 ± 16***  245 ± 53* 95 ± 41   46 ± 10**  219 ± 50* HFD + 25HC 49 ±13   144 ± 24 ## 119 ± 21 ##   188 ± 26 ##   55 ± 12 ##   56 ± 14 #  294 ± 57 ## HFD + 25HC3S 34 ± 9 ##  87 ± 24 # 72 ± 26 # 245 ± 50 76 ±21 47 ± 6 222 ± 54 HCD 57 ± 17**   91 ± 12***  86 ± 10***   250 ± 38**94 ± 14   47 ± 7*** 234 ± 46 HCD + 25HC3S 43 ± 8 ^(†  )  86 ± 7   77 ± 7^(† )  249 ± 39 89 ± 19 44 ± 5 227 ± 48 C57BL/6J female mice were oneach diet for 10 weeks and treated with 25HC or 25HC3S twice and fastedovernight. alues are mean ± SD; n = 15-17; *p < 0.05, **p < 0.01, ***p <0.001, # p < 0.05, ## p < 0.01, ### p < 0.001 compared with HFD-fedvehicle-treated mice. ^(†) p < 0.05 compared with HCD-fedvehicle-treated mice.

A high-performance liquid chromatography (HPLC) analysis of serumlipoprotein profiles showed that 25HC3S treatment did not change LDL,VLDL, and HDL protein levels (FIG. 1A) but markedly decreasedtriglyceride contents in VLDL fractions and slightly decreasedcholesterol contents in HDL fractions (FIG. 1C, E). In contrast, 25HCtreatment significantly increased triglyceride contents in LDL fractionsand cholesterol contents in LDL and HDL fractions (Figures ID, F),consistent with Table 1. These results indicate that administration of25HC3S lowers serum lipid levels by decreasing lipid biosynthesis. Incontrast, administration of 25HC in mice significantly increases serumLDL particles by increasing lipid synthesis and blocking LDL uptake,subsequently increasing cholesterol and triglyceride accumulation inserum.

Analysis of 25HC3S or 25HC in Liver Tissues

To study the effects of 25HC3S or 25HC on hepatic lipid metabolism inHFD-fed mice, the concentration of 25HC3S or 25HC in the liver tissuesfrom treated mice was determined by HPLC analysis. The results indicatedthat 25HC3S or 25HC treatment significantly increased the levels ofthese compounds in liver (FIG. 2). It was observed that a small peak of25HC presented in 25HC3S-treated mice liver (FIG. 2) may indicate asmall part of 25HC3S was degraded to 25HC by STS, which is expressed inthe liver.

Hepatic mRNA Expression in the 25HC3S- or 25HC-Treated Mice

To compare the regulation of lipogenic gene expression in response to25HC3S or 25HC treatment in liver, we determined the mRNA levels by realtime RT-PCR as shown in Table. 2. 25HC3S treatment significantlydecreased the mRNA levels of genes involved in fatty acid biosynthesis.Compared to vehicle-treated mice liver, 25HC3S reduced the mRNA levelsof LXRa, SREBP-1c, ACC1 and FAS by 20%, 45%, 45%, and 70%, respectively.In addition, 25HC3S significantly suppressed ABCA1 expression, which mayexplain the lower level of plasma HDL cholesterol (Table 1 and FIG. 1).In contrast, 25HC basically had the opposite effects: increasing fattyacid synthesis and decreasing fatty acid oxidation (Table 2). 25HCincreased the mRNA levels of SREBP-1c, ACC1, and FAS by 170%, 160% and150%, respectively; decreased the mRNA levels of PPARα, ACOX1 and SCDdecreased by 40%, 55% and 40%, respectively (Table 2). In addition, 25HCincreased SRB1 expression, which could cause an increase in oxidized LDL(oxLDL) uptake and inflammatory responses. These results may explain thehigher levels of serum ALT and AST (Table 1). Interestingly, both 25HCand 25HC3S repressed CYP7α1 expression, which is the rate-limitingenzyme in bile acid synthesis, indicating that 25HC feeds back toinhibit the transcriptional level of CYP7α1. In addition, 25HC decreasedthe mRNA levels of glucokinase (GCK), pyruvate kinase (Pk1r), andglucose 6-phosphatase (G6Pase), but not phosphoenolpyruvatecarboxykinase 1 (PCK1) (Table 2), indicating that the decrease in serumglucose levels is due to the repression of hepatic glycolysis andgluconeogenesis (Table 1). It is interesting that 25HC increasedSULT2B1b expression by 3-fold. This is in contrast to in vitro datawhere 25HC3S suppressed SULT2B1b mRNA level but 25HC has no effect. Thepresent results show that 25HC increases SULT2B1b expression but 25HC3Shas no effect. The mechanism is unknown.

TABLE 2 Relative Hepatic mRNA Expression in the Mice Fed a HFD with 25HCor 25HC3S HFD HFD + 25HC HFD + 25HC3S Fatty acid biosynthesis SREBP-1c1.02 ± 0.10 1.69 ± 0.75*   0.56 ± 0.13** ACC1 1.06 ± 0.27 1.58 ± 0.64*  0.56 ± 0.12** FAS 1.07 ± 0.22 1.45 ± 0.18*   0.33 ± 0.21** LXRα 1.03 ±0.13 0.98 ± 0.09   0.79 ± 0.10* FABP1 0.99 ± 0.23 0.48 ± 0.15** 1.14 ±0.45 FATP 1.10 ± 0.39 0.67 ± 0.12*  1.05 ± 0.29 Fatty acid oxidationPPARα 0.96 ± 0.27 0.59 ± 0.09*  0.93 ± 0.35 ACOX1 0.99 ± 0.23 0.45 ±0.17** 0.82 ± 0.16 SCD 0.93 ± 0.23 0.59 ± 0.13** 0.93 ± 0.27 MCAD 1.04 ±0.29 0.75 ± 0.12   1.36 ± 0.31* CPT1 0.99 ± 0.24 0.94 ± 0.16  1.05 ±0.19 Triglyceride metabolism GPAM 1.09 ± 0.25 1.12 ± 0.27  1.16 ± 0.08MTTP 1.18 ± 0.36 1.04 ± 0.14  1.31 ± 0.20 PLTP 1.01 ± 0.18 0.81 ± 0.36 0.97 ± 0.55 Lipid uptake SRB1 1.04 ± 0.12 1.52 ± 0.31**  1.30 ± 0.22*CD36 1.00 ± 0.27 0.71 ± 0.24*  0.93 ± 0.36 LDLR 0.99 ± 0.06 1.38 ±0.50*  1.07 ± 0.09 Cholesterol efflux ABCA1 1.00 ± 0.18 1.08 ± 0.24  0.71 ± 0.12* ABCG1 1.07 ± 0.50 1.38 ± 0.78  1.14 ± 0.76 Bile acidmetabolism CYP7α 1.08 ± 0.30 0.19 ± 0.18**  0.60 ± 0.18** CYP27α 1.00 ±0.12 1.00 ± 0.12  1.06 ± 0.23 Glucose metabolism G6Pase 0.98 ± 0.34 0.25± 0.13** 1.36 ± 0.77 PCK1 0.98 ± 0.04 1.47 ± 0.37** 1.02 ± 0.25 GCK 1.01± 0.01 0.57 ± 0.16** 1.10 ± 0.68 Pkir 1.10 ± 0.17 0.77 ± 0.13**  0.61 ±0.18* Others SULT2B1b 1.00 ± 0.35 3.33 ± 0.89** 1.29 ± 0.34 Animals weretreated as described in FIG. 1. All values are expressed as the means ±SD; n = 5-6; *p < 0.05, **p < 0.01 compared with HFD mice.

25HC3S Administration Decreases Nuclear SREBP-1 Protein Levels andCytoplasimic FAS and ACC1 Expression in Liver.

SREBP-1c is responsible for up-regulation of fatty acids andtriglyceride biosynthesis by binding to SREBP-1 response elements inresponse genes and increasing expression of the rate-limiting enzymesincluding FAS and ACC1. To determine if 25HC3S inactivated the SREBP-1cpathway, nuclear SREBP-1 protein level in liver was determined byWestern blot analysis. Interestingly, HFD feeding markedly increasednuclear SREBP-1 mature form and induced its target gene ACC1 expression(FIG. 3A-D). 25HC3S significantly suppressed nuclear SREBP-1c,cytoplasmic ACC1 and FAS protein levels by 70%, 50% and 40%,respectively (FIG. 3A-D), which is consistent with mRNA levels as shownin (Table 2). In contrast, 25HC increased nuclear SREBP-1, cytoplasmicACC1 and FAS protein levels (FIG. 3A-D), which could be induced by LXRactivation. These results suggest that 25HC3S treatment suppresseslipogenesis by inhibition of SREBP-1c pathway.

Effects of long-term treatment of 25HC3S on lipid homeostasis in HFD-fedmice

To study the effects of long-term treatment of 25HC3S on lipidhomeostasis, 8-week-old C57BL/6J female mice were fed a HFD for 10weeks, and then, divided into two groups: treated with 25HC3S or vehiclerespectively by peritoneal injection once every three days. During thetreatment, the HFD was continued, and we monitored body mass and caloricintake (FIG. 4A). 25HC3S treated mice stopped increasing body mass whilethe control group kept increasing as shown in FIG. 4B. After 6 weeksinjection, the mice were fasted for 5 hrs, and sacrificed. The liverweight was significantly decreased in 25HC3S treatment group (FIG. 4C).

As expected, 25HC3S significantly decreased plasma cholesterol level ascompared to vehicle-treated mice, but surprisingly, 25HC3S did notsignificantly decrease plasma triglyceride level (data not shown). Liverfunction analysis showed that 25HC3S treatment significantly reducedserum ALK, ALT and AST levels (FIGS. 4D,E,F). These results indicatethat 25HC3S treatment suppresses hepatic inflammation and protects theliver from injury incurred by HFD.

To study the effect of 25HC3S on hepatic lipid metabolism, we measuredhepatic lipid level and gene expression. HFD-fed increased triglyceride,total cholesterol, free cholesterol, and free fatty acid levels in liverby 3-, 3.5-3.2- and 2.5-fold compared to chow-fed mice (p<0.01). Theseincreases were significantly reduced by 30%, 15%, 28% and 23%,respectively, by 25HC3S-treatment (FIG. 5A-D). It was noticed thatcholesterol ester concentration was not affected by 25HC3Sadministration (FIG. 5E). The decrease in lipid level was furtherconfirmed by a liver morphology analysis (FIG. 5F). The livers ofHFD-fed mice were pale and were distended by large cytoplasmic lipidinclusions compared to that of chow-fed mice, suggesting successfulNAFLD model following a HFD feeding. 25HC3S treatment significantlydecreased lipid inclusions.

Gene expression study showed that 25HC3S significantly decreased mRNAlevels of SREBP-1c, ACC1 and FAS by 23%, 41%, and 24%, respectively(Table 3), consistent with the acute treatment (Table 2), but thedifference we found is that 25HC3S long-term treatment significantlydecreased the key enzyme of triglyceride synthesis GPAM (19%), and lipiduptake CD36 (49%).

TABLE 3 Relative Hepatic mRNA Expresison in the Mice Fed a HFD with orwithout 25HC3S HFD HFD + 25HC3S Fatty acid biosynthesis SREBP-1c 0.98 ±0.11  0.76 ± 0.08** ACC1 1.05 ± 0.35 0.63 ± 0.08* FAS 0.96 ± 0.09 0.73 ±0.16* LXRα 1.15 ± 0.17 0.95 ± 0.24  Triglyceride metabolism GPAM 1.02 ±0.16 0.81 ± 0.10* MTTP 1.02 ± 0.22 0.78 ± 0.09* PLTP 0.98 ± 0.32 0.60 ±0.11* Lipid uptake SRB1 1.03 ± 0.18 0.87 ± 0.20  CD36 0.92 ± 0.22  0.47± 0.08** LDLR 1.12 ± 0.23 0.79 ± 0.13  Cholesterol efflux ABCA1 1.01 ±0.11 0.80 ± 0.12* ABCG1 1.09 ± 0.26 0.72 ± 0.19* Inflammatory cytokinesNFκB 1.05 ± 0.34 0.86 ± 0.22  IκBα 1.05 ± 0.25 1.48 ± 0.38  TNFα 1.13 ±0.23 0.60 ± 0.34* IL1α 1.16 ± 0.25  0.54 ± 0.14** IL1β 1.37 ± 0.46 0.58± 0.27* Animals were treated as described in FIG. 4. All values areexpressed as the mean ± SD; n = 5-6 *p < 0.05, **p < 0.01 compared withHFD mice.

Dysregulation of lipid metabolism is frequently associated withinflammatory conditions. 25HC3S treatment significantly suppressed theexpression of TNFα, IL1α, and IL1β, by 47%, 53%, and 58%, respectively(Table 3) where 25HC increased the expression of IL1α and NFκB by 217%and 168%, respectively (data not shown). These results are consistentwith liver function assays showing that 25HC3S suppresses liverinflammatory responses and improves liver damage (FIGS. 4E, F).

DISCUSSION

The present study has shown that the acute treatment of mouse NAFLDmodels with 25HC3S decreases serum lipid levels; and the long term oftreatment decreases both serum and hepatic lipid levels. 25HC3Ssuppresses key gene expressions involved in lipid biosyntheis attranscriptional levels via blocking activation of nuclear receptor LXRsand SREBPs, subsequently suppressing proinflammatory cytokines inducedby HFD. Thus, 25HC3S serves as a potent regulator to reduce hepaticlipid levels effectively.

Example 2. Oxysterol Sulfation by SULT2B1b Suppresses LXR/SREBP-1cSignaling

Pathway and Reduces Serum and Hepatic Lipids in Mouse Models of NAFLD Inthe present study, we further evaluated the effect of SULT2B1b on lipidmetabolism in serum and liver tissues, and possible mechanism in vivo inmouse NAFLD models.

Materials and Methods Animals and Treatment

Eight-week-old female C57BL/6 mice were purchased from Charles RiverLaboratories (Cambridge, Mass.) and LDLR^(−/−) mice were from JacksonLaboratory (Sacramento, Calif.). Mice were hosted under a standard 12/12-hour light/dark cycle. Mice were fed with either standard rodent chowdiet (Harlan Tekiad, Madison, Wis.), or a high-cholesterol diet (HCD,3.1 Kcal/g, 2% cholesterol and 5.7% fat), or a high-fat diet (HFD, 4.5Kcal/g, 0.2% cholesterol and 21.2% fat) for 10 weeks. Mice were theninfected with recombinant adenovirus encoding CMV-driven SULT2B1b (1×10⁸pfu/mouse) through tail vein injection. Ad-CMV-13-Gal adenovirus wasused as control. In addition, some mice were given an intraperitonealinjection of 25HC 2 days after infection with virus as indicated. Micewere sacrificed following an overnight fast 6 days after adenovirusinfection. All protocols were approved by the Institutional Animal Careand Use Committee (IACUC) of the McGuire VA medical center.

Immunohistochemisty

Formalin-fixed liver tissues were processed for histological analysesand stained with hematoxylin and eosin (H&E). Briefly, deparaffinized 4μm sections were stained with rabbit anti-SULT2B1b antibody (AB38412,USA). Immobilized antibodies were detected by theavidin-biotin-peroxidase technique (Vectastain ABC Kits, VectorLaboratories, UK). DAB was used as the chromogen and methyl green orhaematoxylin as the nuclear counterstain.

Lipid levels in liver tissue and sera Liver cholesterol andtriglycerides were extracted and analyzed as previously described inExample 1. Briefly, mouse liver tissue, 100 mg, was homogenized in 1 mlof PBS. The lipids in the homogenates were extracted with 9 ml ofchloroform:methanol (2:1, v/v) overnight, sonicated for 1-2 hrs, andfiltered. The extract, 100 μl, was evaporated to dryness and dissolvedin 100 μl of isopropanol containing 10% Triton X-100 for the cholesterolassay; dissolved in isopropanol for the triglyceride assay; or in NEFAsolution (0.5 g of EDTA-Na₂, 2 g of Triton X-100, 0.76 ml of IN NaOH,and 0.5 g of sodium azide/L of H₂O, pH 6.5) for the free fatty acidsassay. Total and free cholesterol, triglycerides, and free fatty acidsassays were performed according to the manufacturer's instructions.

For serum analysis, the lipid levels and the liver-specific cytosolicenzyme activities of alkaline phosphatase (ALP), alanine transaminase(ALT), and aspartate transaminase (AST) in serum of mice were determinedby clinical biochemistry laboratory blood assays at the VA MedicalCenter. The lipoproteins of cholesterol and triglycerides (VLDL, LDL,and HDL) were measured by gel filtration using high pressure liquidchromatography (HPLC) as described in Example 1 with some modifications.Briefly, serum was centrifuged at 2000 rpm for 2 mm, and 100 μl ofsupernatant was subjected to HPLC with Pharmacia Superose 6HR 10/30column using mobile phase, 154 mM NaCl, 0.1 mM EDTA pH 8.0 at flow rateof 0.2 ml/min. Each fraction was collected starting at 20 min, 1.2min/each (240 μl) for up to 100 min. For cholesterol assay, 180 μl ofeach fraction was transferred to a 96 well plate, and 20 μl Wako totalcholesterol kit 10× reagent buffer was added. After incubating at 37° C.for 3 hrs in the dark, the OD was read at 595 nm. For triglyceridesassay, each 240 μl fraction was evaporated under N₂ gas. The residueswere dissolved in 200 μl of Fisher Scientific Infinity triglyceridereagent, mixed, and transferred to a 96 well plate. The OD value wasread at 500 nm. The protein profile was monitored at OD 280 nm asinternal control.

Analysis of Composition of Oxysterols and Sulfated Oxysterols in LiverTissue

Total lipids in liver tissue were extract by the well-known Folchmethod. Briefly, 200 mg of mouse liver tissue was homogenized in 1 ml ofPBS. 20 ml of chloroform:methanol (2:1, v/v) was added in thehomogenates, sonicated for 1-2 hrs, and filtered, 4 ml of water and 100μl of 1 M K₂CO₃ were added, mixed, and allowed to stand for about 3 hrsfor the phase separation.

The water/methanol (upper) phase, which contains sulfated oxysterols,and chloroform (lower) phase, which contains oxysterols, were evaporatedunder N₂ stream respectively. The residue from water/methanol phase wasre-suspended in 0.5 ml of methanol, 3.5 ml of water and 0.5 ml of NaOH(1 N) by sonication, and the suspension was passed through apreconditioned Sep-Pak tC18 cartridge (Waters, Milford, Mass.) to removenon-sulfated oxysterols. After successively washing the cartridge with 8ml of water, 3.5 ml of 15% acetone and 8 ml of water again, the sulfatedoxysterol fraction was eluted in 5 ml of 75% methanol, which was takento dryness under N2 stream below 40° C. The extracts were thenhydrolyzed in 1 ml of sulfatase (2 mg/ml) at 37° C. for 4 hrs.De-conjugated oxysterols were extracted by Folch's partition(CHCl₃:CH₃OH, 2:1) and the chloroform phase was taken to dryness. Theresidue from chloroform phase was resuspended in 10 ml of hexane andpassed through a pre-conditioned Pep-pak column. The oxysterols wereeluted in 8 ml of 20% isopropanol/hexane after being washed by 3 ml of1% isopropanol/hexane.

The oxysterol samples thus obtained from the methanol/water orchloroform phase were oxidized with cholesterol oxidase. To theoxysterol sample dissolved in 50 μl of 2-propanol were added 450 μl ofwater, 50 μl of 1M potassium phosphate buffer (Kpi) and 1.5 μg ofprogesterone as an internal standard, and the resulting mixture wassonicated for 10 min. To the mixture 0.4 units of cholesterol oxidase in50 μl of Kpi buffer was added and incubated at 37° C. for 1 h. 300 μl ofmethanol was added to stop the reaction and the products, enones, wereextracted 3 times with 2 ml of hexane, and the extracts were evaporatedunder N₂ stream. The residue was re-dissolved in 150 μl of 5%isopropanol in hexane and 100 μl of the solution was subjected to theHPLC as described below.

HPLC analysis was conducted with an Alliance 2695 separation modulefitted with 2487 Dual A absorbance detector (Waters, Milford, Mass.).The separation was carried out on an Ultraspere silica column (5 μm, 4.6mm id×250 mm; Beckman, Urbana, Ill.) and hexane:isopropanol:acetic acid(965:25:10, v:v:v) as an eluent at a flow rate of 1.3 ml/min. The columntemperature was kept constant at 30° C. The enones were monitored at 240nm absorption.

Determination of Gene Expression Involved in Lipid Metabolism

Nuclear and cytosolic proteins from mouse liver tissue were extractedaccording to the manufacturer's instructions. 20 pg nuclear extracts or50 pg cytosolic proteins were loaded on 10% SDS-PAGE for detection ofthe specific proteins, including LXRci, SREBP-1, SREBP-2, ACC-1, FAS,and SULT2B1b, using Lamin Bi and f3-actin as loading control for nuclearand cytosolic proteins respectively. Western blot analysis was performedusing well-known methods, e.g. see Example 1.

Total RNA in liver tissue was isolated by SV total RNA isolation kit(Promega, Wisconsin, WI) following the manufacturer's instructions.Complementary DNA (cDNA) was synthesized retro-transcribing 2 pg oftotal RNA in a total volume of 20 μl using the M-MLV reversetranscriptase (Invitrogen, CA) according to manufacturer's instructions.The relative mRNA levels were measured by quantitative real-timepolymerase chain reaction (qPCR). PCR assays were performed in 96-welloptical reaction plates using the ABI 7500HT machine (AppliedBiosystems, CA). PCR assays were conducted in triplicate wells for eachsample. The following reaction mixture per well was used: 10 μl RT²RealTime™ SYBR Green/Rox PCR master mix (SA Biosciences, MD), 1 μl ofprimer set at the final concentration of 500 nM, 4 μl RNase free water,5 μl cDNA (10 ng). For all experiments the following PCR conditions wereused: denaturation at 95° C. for 10 minutes, followed by 40 cycles at95° C. for 15 seconds, then at 60° C. for 60 seconds. Quantitativenormalization of cDNA in each sample was performed using GAPDH as aninternal control. Suitable primers sequence-based were used.

Statistical analysis was carried out as described in Example 1.

Results Effect of Adenovirus Infection on Liver Toxicity

To optimize the infection condition, the liver toxicity after infectionwith adenovirus was monitored with concentration- and time-dependence.The results showed that ALP, ALT, and AST activities in the serum weresignificantly increased 3, 6, 12, and 24 days after injection withadenovirus (1×10⁹ pfu), and the highest activities were noticed at day 6as shown in FIG. 6A. Consistently, the ratios of liver weight to bodyweight were also significantly increased as shown in FIG. 6B. The serumactivities of ALP, ALT, and AST and the ratios of liver to body weightfollowing injection with different amount adenovirus for 6 days wereshown in FIG. 6C and FIG. 6D. Only 1×0⁹ pfu infection had the higherlevels of ALP, ALT, and AST. Thus, 1×10⁸ pfu for 6 days was selected tostudy the effect of SULT2B1b on lipid metabolism.

SULT2B1b Expression in Different Tissues after Infection withAd-SULT2B1b

Mice were infected with Ad-SULT2B1b or Ad-control through tail veininjection in the condition as described above. Immunohistochemistryanalysis showed that SULT2B1b gene expression was significantlyincreased in liver, aorta and lung tissues, but not in heart or kidneyfollowing infection (FIG. 7A). Consistently, western blot analysisshowed that SULT2B1b gene expression increased by 20 fold in liver, 1.5fold in aorta and 2 fold in lung following Ad-SULT2B1b infection, ascompared to control, while no changes were detected in heart and kidney(FIGS. 7B and C).

Effect of SULT2B1b Overexpression on Lipid Levels in Sera and LiverTissue.

To study the effect of SULT2B1b on the lipid levels, the mice wereinfected by SULT2B1b adenovirus as described above. The serum lipidlevels and specific enzyme activities were measured by clinicallaboratory and are presented in Table 4. Following SULT2B1boverexpression triglyceride levels in serum in C57BL/6 mice fed with HCDwere significantly decreased by 18%, in the presence of 25HC, but nochange in the absence of 25HC as compared to control mice injected with13-Gal virus. In LDLR⁻¹ mice, both serum LDL and HDL were dramaticallyincreased compared to those in C57BL/6 mice. The serum triglyceridelevels were decreased by 32% following SULT2B1b overexpression ascompared to the control in the knockout mice. However, total serumcholesterol levels both in C57BL/6 and LDLR^(−/−) mice were unchangedwith SULT2B1b overexpression. Interestingly, SULT2B1b overexpressiondecreased cholesterol and triglyceride levels in VLDL and LDL fractionin C57BL/6 and LDLR^(−/−) mice while increased their levels in HDLfraction in C57BL/6 mice (FIG. 8A-C). These effects were much strongerin the presence of 25HC (FIG. 8A-C). There is no cytotoxicity followingSULT2B1b overexpression as compared to no infection and 13-Gal virusinfection (Table 4).

TABLE 4 Effect of SULT2B1b overexpression on serum lipids. Wild Type HCDControl SULT Lipids in serum TG (mg/ml) 53 7.3 49 12.4 TC (mg/ml) 11712.7 120 8.4 Glu (mg/ml) 232 24.1 192 26.2* LDL (mg/ml) ND ND ND ND HDL(mg/ml) 90 25.6 99 16.4 Liver function 70 8.6 83 24.8 ALT (IU/L) 40 9.335 7.2 201 34.1 228 49.2 Wild Type HCD + 25HC Control SULT Lipids inserum TG (mg/ml) 39 3.4 32 4.3* TC (mg/ml) 102 8.2 107 9.3 Glu (mg/ml)169 19.1 211 46.2 LDL (mg/ml) ND ND ND ND HDL (mg/ml) 80 10.5 92 11.2Liver function 97 19.2 98 21.1 ALT (IU/L) 77 8.5 88 40.4 229 4.1 26461.9 LDLR^(−/−) Mouse HFD Control SULT Lipids in serum TG (mg/ml) 32892.8 222 47.2* TC (mg/ml) 1338 203.8 1233 155.6 Glu (mg/ml) 222 42.7 26632.3* LDL (mg/ml) 744 84.5 689 94.5 HDL (mg/ml) 529 190.3 497 120.5Liver function 92 23.4 77 19.1 ALT (IU/L) 151 74.7 79 37.4 395 83.9 32978.2 C57BL16 mice and LDLR mice, 8 w, fed with high cholesterol diet(HCD) or high fat diet (HFD) for 10 weeks, then the mice were infectedwith Ad-control orAd-SULT2B1b (1 × 10⁸ pfu) in the presence or absenceof 25HC as indicated. Total cholesterol (TC), triglycerides (TG),glucose (Glu), LDL, HDL and the enzyme activities of ALP, ALT, and ASTin the serum were measured by clinical laboratory. *P < 0.05, **P < 0.01vs. Con. Values are mean SD. ND, not determined.

To determine the effects of SULT2B1b on hepatic lipid levels, totalneutral lipids in liver tissue were extracted by chloroform-methanol(2:1, v/v) mixture. Quantitative analysis showed that overexpression ofSULT2B1b significantly decreased hepatic triglyceride, totalcholesterol, and free cholesterol levels in the presence of 25HC inC57BL/6 mice fed with HCD. No change was detected in the absence of 25HCas compared to control. In LDLR^(−/−) mice, SULT2B1b overexpressionsignificantly decreased total cholesterol and free fatty acids levelsbut did not change triglycerides and free cholesterol levels.Consistently, H&E staining also showed that SULT2B1b overexpressionsubstantially decreased total neutral lipids in liver tissue both inC57BL/6 mice and LDLR mice (FIG. 9A-D).

Effect of SULT2B1b on Oxysterols and Sulfated Oxysterols in LiverTissue.

To study whether generation of sulfated oxysterols is responsible forthe effects of overexpression of SULT2B1b on the serum and hepatic lipidlevels, oxysterols and sulfated oxysterols were exacted from livertissue and analyzed by HPLC. The results showed that SULT2B1boverexpression in the presence of 25HC significantly increased sulfatedoxysterols, especially 25HC3S, and decreased oxysterols, including7-ketocholesterol (7KC), 6β-hydroxycholesterol (60HC), and 25HC (FIG.10A-D). However, in the absence of 25HC, SULT2B1b overexpression did notsignificantly change the levels of oxysterols and sulfated oxysterols(FIG. 10A-D).

Effect of SULT2B1b on Gene Expressions Involved in Lipid Metabolism

To better understand the mechanism of SULT2B1b on lipid metabolism, geneexpressions involved in lipid metabolism were determined. As expected,overexpression of SULT2B1b significantly decreased LXRa and SREBP-1 innuclear protein levels but not SREBP-2 both in C57BL/6 mice andLDLR^(−/−) mice fed with HCD or HFD. Consistently, SULT2B1boverexpression also significantly decreased the cytosolic protein levelsof FAS and ACC1 as shown in FIGS. 11A and B.

Real-time RT-PCR analysis of the gene expressions at mRNA level involvedin lipid metabolism was shown in Table 5. Consistent with proteinlevels, SULT2B1b overexpression significantly decreased mRNA levels ofLXRa, SREBP-1, SREBP-2, GPAM, ACAT2, CYP27A, ABCA1, ABCGI and STS in themice injected peritoneally with 25HC; in the absence of 25HC, SULT2B1boverexpression only decreased mRNA levels of LXRa, SREBP-1, and ABCA1.

TABLE 5 Effect of SULT2BI b overexpressoin on gene expressions involvedin lipids metabolism at mRNA level. Wild Type HCD Control SULT Fattyacid metabolism SREBP-1c 1.0 0.29 0.57 0.19* ACC1 1.0 0.37 0.93 0.15 FAS1.0 0.41 0.76 0.21 LXRα 1.0 0.14 0.81 0.09* PPARgamma 1.0 0.26 0.59 0.17FABP4 1.0 0.51 0.68 0.19 FATP 1.0 0.39 0.59 0.15* Triglyceridemetabolism GPAM 1.0 0.62 0.94 0.36 MTTP 1.0 0.55 0.93 0.41 PLTP 1.0 0.690.94 0.51 Cholesterol metabolism SREBP-2 1.0 0.68 0.92 0.50 HMGR 1.00.19 0.90 0.08 LDLR 1.0 0.20 1.13 0.25 ACAT1 1.0 0.12 1.02 0.35 ACAT21.0 0.27 0.61 0.10* Cholesterol efflux ABCA1 1.0 0.22 0.65 0.09* ASCG11.0 0.23 1.15 0.28 Bile acid metabolism CYP7a 1.0 0.33 0.78 0.42 CYP27a1.0 0.27 0.80 0.14 Others SILT2B1b 1.0 0.24 193.5 46.1** STS 1.0 0.730.52 0.23 Wild Type HCD + 25HC Control SULT Fatty acid metabolismSREBP-1c 1.0 0.16 0.53 0.18* ACC1 1.0 0.27 0.79 0.09 FAS 1.0 0.50 0.570.14 LXRα 1.0 0.30 0.63 0.08* PPARgamma 1.0 0.29 0.58 0.15* FABP4 1.00.45 0.43 0.16* FATP 1.0 0.25 0.62 0.17* Triglyceride metabolism GPAM1.0 0.33 0.69 0.11* MTTP 1.0 0.28 0.81 0.16 PLTP 1.0 0.13 0.85 0.20Cholesterol metabolism SREBP-2 1.0 0.08 0.71 0.07** HMGR 1.0 0.24 0.960.24 LDLR 1.0 0.39 0.53 0.11* ACAT1 1.0 0.18 1.11 0.29 ACAT2 1.0 0.210.73 0.06* Cholesterol efflux ABCA1 1.0 0.22 0.65 0.16* ASCG1 1.0 0.231.15 0.22* Bile acid metabolism CYP7a 1.0 0.33 0.78 0.31 CYP27a 1.0 0.270.80 0.07* Others SILT2B1b 1.0 0.24 193.5 114.8** STS 1.0 0.73 0.520.06* LDL^(−/−) Mouse HFD Control SULT Fatty acid metabolism SREBP-1c1.0 0.23 1.14 0.27 ACC1 1.0 0.22 0.84 0.22 FAS 1.0 0.27 1.01 0.20 LXRα1.0 0.19 0.82 0.11* PPARgamma 1.0 0.27 0.81 0.19* FABP4 1.0 0.27 0.870.27 FATP 1.0 0.33 0.51 0.18** Triglyceride metabolism GPAM 1.0 0.190.90 0.17 MTTP 1.0 0.17 0.89 0.16 PLTP 1.0 0.15 0.69 0.11** Cholesterolmetabolism SREBP-2 1.0 0.14 0.94 0.16 HMGR 1.0 0.26 0.91 0.29 LDLR 1.00.34 0.80 0.22 ACAT1 1.0 0.23 1.00 0.13 ACAT2 1.0 0.19 0.89 0.10Cholesterol efflux ABCA1 1.0 0.26 0.89 0.17 ASCG1 1.0 0.27 0.83 0.19Bile acid metabolism CYP7a 1.0 0.20 0.90 0.48 CYP27a 1.0 0.21 1.10 0.21Others SILT2B1b 1.0 0.44 324.6 36.7** STS 1.0 0.57 1.28 0.37 C57BL16mice and LDLR^(−/−) mice, 8 w, fed with high cholesterol diet (HCD) orhigh fat diet (HFD) for 10 weeks, then the mice were infected withAdcontrol or Ad-SULT2B1b (1 × 10⁸ pfu) in the presence or absence of25HC as indicated. Gene expressions involved in lipid metabolism wereanalyzed by real-time PCR at mRNA level. *P < 0.05, **P < 0.01 vs. Con.

DISCUSSION

The present study shows that SULT2B1b overexpression increases 25HCsulfation and its product 25HC3S mainly in liver tissue in vivo in mouseNAFLD animal models. Subsequently, SULT2B1b decreases serum and hepatictriglycerides, total cholesterol, free cholesterol, and free fattyacids, accompanied by reduction in key regulators and enzymes in lipidmetabolism, including SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, andfatty acid synthase. The results confirm that oxysterol sulfation bySULT2B1b plays an important role in lipid metabolism in vivo, indicatingthat oxysterol sulfation is another systematic signaling pathwayinvolved in lipid metabolism. These findings also suggest that thesulfation product, 25HC3S, is an important endogenous regulator of lipidbiosynthesis and has a potential to serve as a new medicine for therapyof NAFLD. These results indicate that the sulfated oxysterol may act asan LXR antagonist rather than only an inactive form of LXR ligand andthat SULT2B1b plays an important role in lipid homeostasis, determiningthe balance between 25HC and 25HC3S, and represents a novel target forlipid metabolic disorder related diseases, including NAFLD andatherosclerosis.

In summary, the results indicate that 25HC sulfation by SULT2B1bsubstantially decreases serum and hepatic lipid levels via inhibitingthe LXR-SREBP-1c signaling pathway. This finding supports the hypothesisthat 25HC3S is an important endogenous regulator of lipid biosynthesis.This pathway represents a novel target for pharmacological interventionin NAFLD and other lipid-related disorders.

Example 3. Cholesterol Metabolite, 5-Cholesten-3Beta, 25Diol 3-Sulfate(25HC3S) Promotes Hepatic Proliferation in Mice

25HC3S is a sulfated oxysterol present in the nuclei of hepatocytes,where it plays an important role in regulating lipid metabolism In thepresent Example, we have shown for the first time that 25HC3Ssubstantially up-regulates proliferative gene expression and induces DNAreplication in liver. Conversely, treating mice with theliver-X-receptors (LXR) synthetic agonist, T0901317, leads to effectivesuppression of 25HC3S-induced proliferation, indicating the involvementof LXR signaling. These findings provide a previously undescribedfunction of 25HC3S in liver, and may shed light on our understanding ofproliferative mechanisms regulated by the acidic pathway of bile acidbiosynthesis in liver.

EXPERIMENTAL Synthesis of 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S)

25HC3S was synthesized as described in Example 1, with somemodifications. Briefly, a mixture of 25-hydroxycholesterol (402 mg, 1mmol) and triethylamine-sulfur trioxide pyridine complex (106 mg, 1mmol) in 5 ml of dry pyridine was stirred at 25° C. for 2 h. After thesolvents were evaporated at reduced pressure, products were purified byHPLC using a silica gel column, methylene chloride and methanol (5%)were used as the mobile phase. The product was further purified byreverse-phase HPLC using C18 column as obtained as a white powder. Thestructure of the product was characterized by mass spectrum and nuclearmagnetic resonance spectroscopy analysis.

Synthesis of [³H]-25HC3S

A mixture of [³H]-25HC (10 μCi), cholesterol (4 mg) andtriethylamine-sulfur trioxide complex (1 mg) in 500 ul of dry pyridinewas stirred at 20° C. for 1h. After the solvents were evaporated, 1 mlof alkaline methanol (pH 8) was added, mixed, filtered, and purified byHPLC (mobile phase A is 20% CH₃CN in HO, B is 20% CHCN in CH₃OH, 0-20min, A 50%-0%, B 50%-100%, 2 ml/min; 20-35 min, A 0%, B 100%, 2 ml/min;35-40 min, A 0%-50%, B 100%-50%, 2 ml/min. A pure peak with the sameretention time as standard 25HC3S and with high radioactivity wascollected for further usage.

Animal Maintenance and Treatment.

Nine- to 12-week-old C57BL/6 mice were used and received humane care inaccordance with the institutional guidelines and the National Institutesof Health Guide for the Care and Use of Laboratory Animals. Mice wererandomly allocated into the following groups (n=3-5/group): (i) vehiclegroup-mice were administrated with 10% ethanol in phosphate buffersolution (PBS) intravenously (i.v.); (ii) 25HC3S group-same as vehiclegroup but in which 25HC3S (5 mg/kg, 10% ethanol) was administered i.v.;(iii) T0901317 group-identical to 25HC3S group except the administrationof T0901317 (5 mg/kg, 10% ethanol administered i.v.; Cayman Chemical,Ann Arbor, Mich.); (iv) 25HC3S+T0901317 group-mice were administratedwith 25HC3S and T0901317 (5 mg/kg, 10% ethanol administered i.v.). Toget the mouse model with high endogenous 25HC3S, mice were received ani.v. injection of adenovirus encoding human SULT2B1b (Ad-SULT2B 1 b,1×10⁸ pfu/mouse), and further supplemented with 25HC (25 mg/kg, 10%ethanol; Research Plus, Inc. Bayonne, N.J.) intraperitoneally (i.p.), 2days after the adenovirus infection and followed by once every two days[Bai Q, et al. Oxysterol sulfation by cytosolic sulfotranseferasesuppresses liver X receptor/sterol regulatory element binding protein-1csignaling pathway and reduces serum and hepatic lipids in mouse modelsof nonalcohol fatty liver disease. Metabolism. (Accepted)]. Adenovirusencoding β-Gal (Ad-Control) or 10% ethanol (vehicle) in PBS was used ascontrols, respectively. Animals were sacrificed at 48 h afteradministration or day 5 following adenovirus injection. Blood sampleswere collected at sacrifice and serum was separated, stored at −80° C.until assayed. Alanine aminotrasferase (ALT), aspartate aminotransferase(AST), and alkaline phosphatase (AP) concentrations were measured in theclinical lab at the McGuire VA Medical center. Liver tissues wereharvested and divided into two portions, one for analysis of geneexpression and the other for morphological studies.

Biodistribution and Pharmacokinetic Studies.

In a separate set of experiments, mice received an i.v. injection of[³H]-25HC3S (1×10⁶ cpm/mouse) and 25HC3S (5 mg/kg). At 4, 24, 48, and 96h after the injection, mice were sacrificed, tissues (brain, heart,lung, liver, kidney, spleen, stomach, small intestine, skeletal muscle,and colon) were rapidly dissected and weighed, and the radioactivity wasmeasured using a liquid scintillation counter with automatic decaycorrection (Beckman Counter, Atlanta, Ga.). Tissue values werecalculated as a percentage of injected counts per gram of tissue (%IC/g). For pharmacokinetics, blood samples (30 μl) were collected atselected times after injection from 0 to 96 h via tail clip. Counts ofradioactivity were expressed as IC/ml of blood.

RNA Extraction.

Total liver RNA was extracted using SV Total RNA Isolation Kit (Promega,Wisconsin, WI) according to the manufacture's instruction. RNA puritywas checked by spectrophotometer. Complementary DNA (cDNA) wassynthesized by retrotranscribing 2 ug of total RNA with Oligo dT primersand M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, Calif.).

Quantitative Real-Time Polymerase Chain Reaction (RTqPCR) and Cell CycleRT2 Profiler™ PCR Array.

PCR assays were performed in 96-well optical reaction plates using theABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City,Calif.). PCR assays were conducted in triplicate wells for each sample.Baseline values of amplification plots were set automatically andthreshold values were kept constant to obtain normalized cycle times andlinear regression data. The following reaction mixture per well wasused: 2×SYBR Green PCR Master Mix (10 ul), primer at the finalconcentration of 10 uM (1 ul), RNAse-free water (4 ul), cDNA (5 ul, 10ng). For all experiments the following PCR conditions were used:denaturation at 95° C. for 10 minutes, followed by 40 cycles at 95° C.for 15 seconds, then at 60° C. for 60 seconds. Quantitativenormalization of cDNA in each sample was performed using 18S gene as aninternal control. Relative quantification was performed using the ΔΔCTmethod. Suitable primers were used for RtqPCR.

The real-time PCR array was performed using the Mouse Cell cycle RT2Profiler™ PCR Array consisting of 84 key genes involved in apoptosis andproliferation as the manufacturer's instruction (SABiosciences,Frederick, Md.). Total mRNA was reverse-transcribed to cDNA as describedabove. Three of biologically-distinct specimens were pooled to reducevariance. The cDNA was then amplified precisely like real-time PCR inthe primers-preloaded PCR plates on the ABI 7500 Fast Real-Time PCRSystem. ΔΔCT values between different stimulated samples were analyzedas the manufacturer's instruction, which output p-values forfold-regulation change. Only those genes whose expression levels showeda 1.5-fold or greater expression difference between groups wereconsidered significant changes between groups.

Liver Histology.

Liver specimens collected at sacrifice were fixed in 10%neutral-buffered formalin, embedded in paraffin, sectioned at 4 um,deparaffinized, and rehydrated. Endogenous peroxidase was inactivated byincubation in 3% hydrogen peroxide in absolute methanol for 30 min.Antigen retrieval was performed by microwaving of the sections incitrate buffer (PH 6.0) for 10 min. Sections were incubated overnight at40° C. with the primary monoclonal antibody against proliferating cellnuclear antigen (PCNA) (ab29, Abcam, Cambridge, Mass.). After washingwith PBS, immobilized antibodies were detected by theavidin-biotin-peroxidase complex technique (Vectastain@ ABC Kit, VectorLaboratories, Burlingame, Calif.). DAB (Vector Laboratories, Burlingame,Calif.) and haematoxylin (Sigma-Aldrich, St. Louis, Mo.) was used as thechromogen and nuclear counterstain, respectively. PCNA-positive andPCNA-negative nuclei were counted in five randomly selected fields foreach sample, and each group included sections from at least 3 mice. Thequantitation of PCNA expression was expressed as the PCNA labelingindex.

Western Blot Analysis.

Cell lysates were prepared from frozen mouse liver tissues. Totalproteins, 100 rig, were separated by 10% SDS-PAGE, and transferred toPVDF membrane (Millipore, Eschborn, Germany). Specific protein wasprobed with specific antibodies against PCNA, human SULT2B1b (Santa CruzBiotechnology, Santa Cruz, Calif.), LXRa (Abcam, Cambridge, Mass.),ATP-binding cassette transporters 1 (ABCA1) (Abcam, Cambridge, Mass.),and SREBP-1c (Santa Cruz Biotechnology, Santa Cruz, Calif.). 13-Actin(Sigma-Aldrich, St. Louis, Mo.) was used as a loading control. Theimmunoreactive bands were detected by Fujifilm Medical System (Fujifilm,Stamford, Conn.) and quantified by Advanced Image Data Analyzer (AidaInc., Straubenhardt, Germany).

Statistical Analysis.

Statistical analyses were carried out as described in Example 1.

Results Pharmacokinetics and Tissue Biodistribution.

³H-Radioactivity counts in the blood after i.v. injection of [³H]-25HC3Sand 25HC3S was shown in FIG. 12A. The compound showed slow clearance.³H-Radioactivity in blood began to increase at 0.25 h, reached themaximum level of 7% IC/g at 1 h, and decreased to the half level at 48h.

The tissue biodistribution of [³H]-25HC3S were measured at 4, 24, 48 and96 h after the i.v. administration. As shown in FIG. 12B, most of theorgans exhibited the highest uptake of ³H-radioactivity at 4 h.Radioactivity remained at the level until 24 h, and gradually decreasedwith time. No obvious trend of radioactivity distribution was observedamong spleen, liver, kidney, lung, small intestine, and colon. Theradioactivity in these organs was relatively higher than those in heart,muscle, and brain at each time point after the injection. The results oflong half life and wide distribution indicate that no specificreceptor(s) of 25HC3S presents in vivo cells and tissues.

Effect of 25HC3S on Proliferative Gene Expression in Mouse LiverTissues.

In order to investigate the effect of 25HC3S on the hepaticproliferation, 48 h (half-decay period) was chosen in this study. Micewere treated for 48 h with different concentrations of 25HC3S (0-10mg/kg). ALT, AST and AP activities were determined in mouse serumfollowing the administration. ALT, AST and AP in serum of mice with25HC3S administration were slightly higher than those without treatment.However, no significant differences were seen among the four groups(data not shown), indicating no toxicity where 25HC3S was administrated.The hepatic mRNA levels of genes related to cell cycle progression,including cMyc, cyclin A, forkhead Box m1b (FoxM1b), and its target genecell division cycle 25b (CDC25b), which control the cell cycleprogression through GU/S and G2/M phases were investigated following theadministration. RTqPCR analysis showed that at doses of 25HC3S lowerthan 5 mg/kg, mouse liver tissues displayed a significant up-regulationin the expression of these genes, and in a 25HC3S dose-dependent manner(FIG. 13A-D). However, when the concentration of 25HC3S reached to 10mg/kg, most of these gene (except cMyc) expressions recovered to thenormal level.

25HC3S Regulates the Expression of Apoptotic and Cell Cycle RelatedGenes.

To elicit the effects of 25HC3S on liver proliferation and to identifythe genes regulated by 25HC3S in promoting proliferation, a real-timePCR array encompassing 84 genes associated with apoptosis and cell cycleprogression was used. Mice were treated with vehicle or 25HC3S (5 mg/kg)for 48 h. Three of biologically-distinct mRNA samples were pooled toreduce variance, and the real-time array was run for each treatment.Table 6 lists the genes that were regulated by 1.5-fold difference orgreater compared to vehicle group. Treatment with 25HC3S resulted in 18genes up-regulated and 6 genes down-regulated. Interestingly, most ofthe up-regulated genes were positively related to the regulation of cellcycle progression, anti-apoptosis, and cell differentiation. 25HC3Ssubstantially increased the expression of Wt1 (3.9-fold), an oncogeneinvolved in the cell differentiation and viability, and Pcna (3.7-fold)encoding PCNA protein serves as a proliferative cell marker; andsignificantly increased the expression of Ccne2 encoding cyclin E2, anessential regulator for the cell cycle at the late G1 and early S phase,and Ccnb2 encoding cyclin B2, also serves as a proliferation marker andis crucial for the control of cell cycle at the G2/M transition. On theother hand, 25HC3S substantially decreased the expression of Chek2 by4.5-fold encoding CHK2, an important protein kinase involved in cellcycle arrest in response to DNA damage and Apaf1 by 3-fold encodingapoptotic peptidase activating factor 1, a cytoplasmic protein thatinitiates apoptosis. Taken together, these data demonstrate that 25HC3Smay enhance liver proliferation potential by up-regulating proliferativegene expression and down-regulating apoptosis gene expression.

TABLE 6 Mouse Profiler ™ Gene Expression Array analysis of mouse livertissues induced by 25HC3S. Differential gene expression in response to25HC3S treatment (5 mg/kg, 2 days) versus vehicle treatment was analyzedusing the Mouse Cell cycle RT² Profiler ™ PCR Array. Threebiologically-distinct mRNA samples were pooled to reduce variance. Genesequally down- or up-regulated by at least 1.5-fold in response to 25HC3Streatment were shown. Fold Functional Gene Grouping Gene Symbol changeGene Name Regulation of Cell cycle and Ccnb2(NM_007630) 1.90 Cyclin B2Genes Related to the Cell Ccne2(NM_009830) 2.52 Cyclin E2 CycleCdc25a(NM_007658) 1.53 Cell division cycle 25 homolog A (S. pombe)Cdc25c(NM_009860) 1.56 Cell division cycle 25 homolog C (S. pombe)Pcna(NM_011045) 3.77 Proliferating cell nuclear antigen E2f3(NM_010093)2.0193 E2F transcription factor 3 Esr1(NM_007956) 2.2535 Estrogenreceptor 1 (alpha) Mdm2(NM_010786) 1.6222 Transformed mouse 3T3 celldouble minute 2 Wt1(NM_144783) 3.90 Wilms tumor 1 homolog Cell CycleArrest and Cdkn1a(NM_007669) 3.0411 Cyclin-dependent kinase NegativeRegulation of Cell inhibitor 1A (P21) Cycle Gadd45a(NM_007836) −1.59Growth arrest and DNA-damage-inducible 45 alpha Brca1(NM_009764) −1.87Breast cancer 1 Chek2(NM_016681) −4.65 CHK2 checkpoint homolog (S.pombe) Trp73(NM_011642) 2.5194 Transformation related protein 73 CellDifferentiation Hif1a(NM_031168) 1.7576 Hypoxia inducible factor 1,alpha subunit Myod1(NM_010866) 1.56 Myogenic differentiation 1Nf1(NM_010897) 2.10 Neurofibromatosis 1 Induction of ApoptosisApaf1(NM_009684) −3.21 Apoptotic peptidase activating factor 1Tnf(NM_013693) −2.76 Tumor necrosis factor Tnfrsf10b(NM_020275) −2.28Tumor necrosis factor receptor superfamily, member 10b Prkca(NM_011101)2.29 Protein kinase C, alpha Anti-apoptosis Bag1(NM_009736) 2.0488Bcl2-associated athanogene 1 Bnip3(NM_009760) 2.0112 BCL2/adenovirus E1Binteracting protein 3 DNA Repair Atr(NM_019864) −2.15 Ataxiatelangiectasia and rad3 related Xrcc5(NM_009533) −1.90 X-ray repaircomplementing defective repair in Chinese hamster cells 5 TranscriptionFactors Ep300(NM_177821) 1.8114 E1A binding protein p300

Induction of DNA Replication by Exogenous 25HC3S in Mouse Liver Tissues.

Proliferation in liver was examined with liver histology by countingPCNA labeling index. FIG. 14B shows PCNA staining in a liver specimencollected from 25HC3S treated mice, compared to vehicle control (FIG.14A). Immunoreaction was observed as strong reaction (short arrow) orweak reaction (long arrow). The labeling index of cells with nuclearreaction was 12% in this case. We used this standard to evaluate allsections. Immunohistochemical analysis showed that treatment of micewith 25HC3S (5 mg/kg) for 48 h significantly increased the PCNA labelingindex in liver as compared to the vehicle group (FIG. 14C). While nosignificant differences were seen between the vehicle group andno-treatment group (data not shown). The results indicate that 25HC3Spromotes mouse liver proliferation. Induction ofDNA replication byendogenous 25HC3S in mouse livers. SULT2B1b is responsible for thesynthesis of 25HC3S from 25HC. SULT2B1b overexpression in the presenceof 25HC can effectively elevate 25HC3S levels in both primaryhepatocytes and mouse liver. To further confirm the proliferationinduction by endogenous 25HC3S in liver, PCNA positive cells werecompared following overexpression of SULT2B1b. As theimmunohistochemical analysis shown in FIGS. 15A and B, SULT2B1b resultedin a significant increase in the liver PCNA labeling index at day 5following Ad-SULT2B 1 b infection with no evidence of toxicity (data notshown). The PCNA labeling index induced by SULT2B1b was furtherincreased (up to 30%) in the livers of mice supplemented with 25HC.These results confirm that 25HC3S promotes liver proliferation.

Effect of 25HC3S on LXR Activity and its Target Gene Expression in theMouse Liver Tissues.

To understand the possible mechanisms by which 25HC3S promotes hepaticproliferation, the expression of genes regulated by LXR signalingpathway was analyzed in mouse liver tissues. As shown in FIGS. 16A andB, injection of mice with exogenous 25HC3S (5 mg/kg, 48 h) inhibited theactivity of LXR response in liver, the protein levels of LXRa and itstarget genes ABCA1 and SREBP-1c in the 25HC3S group decreased by 40%,50%, and 30%, respectively. For PCNA, the exogenous 25HC3S increased itsprotein level by 2-fold. Similar down-regulated liver LXR signaling andup-regulated PCNA expression were also observed in Ad-SULT2B 1 binfected mouse, where SULT2B1b overexpression in the presence (orabsence) of 25HC decreased the protein levels of LXRa by 70% (60%), andSREBP-1c by 43% (25%); and increased the protein level of PCNA by 2-fold(2.5-fold), as compared to the Ad-control group (FIGS. 17A and B). Inthe Ad-Control and 25HC co-treatment group, 25HC effectivelyup-regulated the expression of ABCA 1 and SREBP-1c, indicating theactivation of LXRs signaling (FIGS. 17A and B). The data not only areconsistent with previous studies showing the inactive effect of 25HC3Son LXR response in primary hepatocytes, but also support the idea that25HC is a natural LXR ligand in vivo. Considering that LXR activationinduces growth arrest and inhibits proliferation in many cells andanimal models, the stimulation of proliferation by 25HC3S is related tothe inactivation of LXR signaling.

Role of LXR Pathway on 25HC3S-Mediated Proliferation in Mouse Livers.

T0901317 is a potent synthetic LXR agonist. As an additional approach toinvestigate whether LXR signaling repression plays a role in25HC3S-induced proliferation, we detected the effect of i.v.administration of T0901317 at 5 mg/kg to mice. As shown in western blotanalysis in FIGS. 18A and B, T0901317 administration induced, at day 2after the injection, the expressions of LXR target genes ABCA1 andSREBP-1c in both the presence and absence of 25HC3S as compared to thevehicle group. Furthermore, the T0901317 administration significantlyrepressed the 25HC3S-induced PCNA expression. The results furtherconfirm that 25HC3S induces proliferation via inactivation of LXRsignaling.

DISCUSSION

The present study shows that LXR signaling pathway is down-regulated inlivers of mice treated with 25HC3S). Our data showed that the presenceof exogenous 25HC3S significantly increases the expression ofproliferative genes at doses lower than 5 mg/kg. However, this effect isnot observed when the concentration reaches to 10 mg/kg. In ourhigh-performance liquid chromatography studies, we detected a small peakof 25HC in 25HC3S-treated mice liver. Thus, it is possible that thereduced effect of 25HC3S on proliferation is related to the accumulationof 25HC.

Example 4. Cytosolic Sulfotransferase 2B1b Promotes HepatocyteProliferation Gene Expression In Vitro and In Vivo

In the present Example, we show for the first time that overexpressionof SULT2B1b promotes hepatic proliferation. Decreases of SULT2B1bexpression in primary rat hepatocytes (PRH) significantly repress theexpression of cell cycle regulatory genes, including forkhead Box m1b(FoxM1b), cyclin-dependent kinase 2 (CDK2), and Cyclin A. Notably, LXRactivation by T0901317 in cultured PRH and mouse liver effectivelysuppresses SULT2B1b-induced proliferation, indicating that the promotionof proliferation by SULT2B1b is via LXR signaling. These findings shedlight on a previously undescribed role of SULT2B1b in enhancing liverproliferation.

Materials and Methods Animals and Treatment.

Nine- to 10-week-old C57BL/6 mice were used and received humane care inaccordance with the institutional guidelines and the National Institutesof Health Guide for the Care and Use of Laboratory Animals. For vectoradministration, the vector in 100 ul of sterile phosphate-bufferedsaline (PBS) was infused through tail vein. Two groups were used(n=3-5/group): the control group (Con) received 1×10⁸ pfu/mouse ofadenovirus encoding 13-Gal (Ad-Control), while the SULT2B1b group (Sult)received a simultaneous injection of 1×10⁸ pfu/mouse of adenovirusencoding human SULT2B1b (Ad-SULT2B 1 b).

For study of LXR signaling pathway, mice were given intraperitonealinjections of T0901317 (25 mg/kg; New Cayman Chemical, Ann Arbor, Mich.)or 25HC (25 mg/kg; Research Plus, Inc. Bayonne, N.J.) 1 h after vectoradministration, and followed by once every two days. Animals weresacrificed at day 0, 2, 4, 5, and 12 after vector administration. Bloodsamples were collected at sacrifice and serum was separated, stored at−80° C. until assayed. Alanine aminotrasferase (ALT), aspartateaminotransferase (AST), and alkaline phosphatase (AP) concentrationswere measured in the clinical lab of the McGuire VA Medical center.Liver tissues were harvested and divided into two portions, one for geneexpression analyses and the other for morphological studies.

Immunohistochemistry.

Liver specimens collected at sacrifice were fixed in 10%neutral-buffered formalin, embedded in paraffin, sectioned at 4-μm,deparaffinized and rehydrated. Endogenous peroxidase was inactivated byincubation in 3% hydrogen peroxide in absolute methanol for 30 min.Antigen retrieval was performed by microwaving of the sections incitrate buffer (pH 6.0) for 10 min. Sections were incubated overnight at4° C. with the primary monoclonal antibody against proliferating cellnuclear antigen (PCNA) (ab29, Abcam, Cambridge, Mass.). After washingwith PBS, immobilized antibodies were detected by theavidin-biotin-peroxidase technique (Vectastain@ ABC Kit, VectorLaboratories, Burlingame, Calif.). DAB (Vector Laboratories, Burlingame,Calif.) and haematoxylin (Sigma-Aldrich, St. Louis, Mo.) was used as thechromogen and nuclear counterstain, respectively. The primary antibodywas omitted as negative control. PCNA-positive and PCNA-negative nucleiwere counted in five randomly selected fields for each sample, and eachgroup included sections from at least 3 mice. The quantitation of PCNAexpression was expressed as the percentage of PCNA-positive cells.

Double Immunofluorescent Staining.

Liver tissues from the mice at day 4 after Ad-SULT2B 1 b infection wereused for double immunofluorescence to locate the expression of PCNA andhuman SULT2B1b. Deparaffinized 4-μm sections were cultured with acocktail mix of two primary antibodies: mouse anti-PCNA antibody, 1:800(ab29, Abcam, Cambridge, Mass.) and rabbit anti-SULT2B1b antibody, 1:30(ab38412, Abcam, Cambridge, Mass.). Subsequent antibody detection wascarried out with secondary antibodies: Alexa Fluor 488 goat anti-mouseIgG, 1:500 (Invitrogen, Carlsbad, Calif.) and Alexa Fluor 488 goatanti-rabbit IgG, 1:500 (Invitrogen, Carlsbad, Calif.). Negative controlwas performed by replacing the primary antibody with PBS. Sections wereexamined with a fluorescence microscope and merged images were formedusing Adobe Photoshop CS2.

Culture of PRH.

PRH were prepared as previously described (24). Briefly, parenchymalcells (3.5×10⁶) were inoculated in 60-mm plastic Petri dishes coatedwith rat tail collagen. The plates contained 3 ml of Williams E culturemedia supplemented with thyroxine (1 μM), dexamethasone (0.1 μM), andpenicillin (100 units/ml). Three hours after plating, culture medium wasremoved and fresh medium was added. Cells were then infected withAd-SULT2B1b at a multiplicity (MOI) of 10 plaque forming units/cell(pfu/cell) or 100 nM of siRNA-SULT2B1b (ON-TARGET plus siRNA of ratSULT2B1b, Thermo Scientific Dharmacon, Lafayette, Colo.) as previouslydescribed (31). The Ad-Control and siRNA-Control (ON-TARGET plusnegative control siRNA) were used as controls, respectively. Twenty fourhours after infection, cells were treated with T0901317 (1.5 μM) or 25HC(3 μM) for another 24 hours. The cells were harvested at the timepointsas indicated in the text.

Cell Survival Assay.

Cultured hepatocyte-viability following treatments with Ad-SULT2B1b orsiRNA-SULT2B1b for 24 h to 48 h, was evaluated using Cell ProliferationKit I (Roche, Indianapolis, Ind.) according to the manufacturer'sprotocol. Briefly, cultured cells were treated with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4h and then subcultured in solubilization solution. The absorbance ofmodified dye was measured at 570 nm. Assays were performed intriplicates. Ad-Control, siRNA-Control, and no treatment were used ascontrols.

Western Blot.

Cell lysates were prepared from frozen mouse liver tissues or PRH, and50 μg of the total proteins were separated by 10% SDS-PAGE, transferredto polyvinylidene difluoride membrane (Millipore, Eschborn, Germany).Specific protein was probed with specific antibodies against PCNA, humanSULT2B1b (Santa Cruz Biotechnology, Santa Cruz, Calif.), LXRa (Abcam,Cambridge, Mass.), ATP-binding cassette transporters 1 (ABCA1) (Abcam,Cambridge, Mass.), and SREBP1 (Santa Cruz Biotechnology, Santa Cruz,Calif.). 1-Actin (Sigma-Aldrich, St. Louis, Mo.) was used as a loadingcontrol. Western analysis was performed by methods known in the art.

Reverse-Transcription Polymerase Chain Reaction (RT-PCR).

Total RNAs were isolated from mouse livers and PRH with SV Total RNAIsolation Kit (Promega, Wisconsin, WI). Two micrograms of total RNA wasused for first-strand cDNA synthesis as recommended by the manufacturer(Invitrogen, Carlsbad, Calif.). Chosen genes were amplified by PCR. ThePCR fragments were visualized on a 1.5% agarose gel containing 5 mg/mlethidium bromide. Quantitative real-time PCR (RTqPCR). RTqPCR wasperformed using SYBR Green (Invitrogen, Carlsbad, Calif.) on theABI-7500 Fast Real-Time PCR System (Applied Biosystems, Foster City,Calif.). The final reaction mixture contained 10 ng of cDNA, 100 nM ofeach primer, 10 μg of 2×SYBR Green PCR Master Mix, and RNase-free waterto complete the reaction mixture volume to 20 μl. All reactions wereperformed in triplicate. The PCR was performed with a hot-startdenaturation step at 95° C. for 10 min and then was carried out for 40cycles at 95° C. for 15 s and 60° C. for 1 min. The intensity offluorescence was read during the reaction, allowing a continuousmonitoring of the amount of PCR product. The data were normalized tointernal controls, 18S gene or GAPDH. Suitable primers were used for PCRand RTqPCR.

Statistical Analysis.

Data are expressed as mean±standard deviation (SD). Statisticalcomparison was made using Student's t test for unpaired samples. A valueof P<0.05 was defined as statistical significant.

Results Overexpression of SULT2B1b In Vivo by a Recombinant Adenovirus.

ALT, AST and AP activities were determined in mouse serum followingadenovirus infection. ALT, AST and AP in serum of mice with Ad-Controlor Ad-SULT2B 1 b injection were slightly higher than those withoutvirus. However, no significant differences were seen among the threegroups (data not shown), indicating no toxicity was observed where theadenovirus was infected. RT-PCR and western blot analysis of humanSULT2B1b expression in the livers at day 0, 2, 4, 5 and 12 afterinjection were performed to verify the transgene expression. HumanSULT2B1b mRNA and protein levels were substantially increased in theliver following infection of mice with Ad-SULT2B1b (FIGS. 19A and B):started the increase at day 2, increased to its highest level at day 4and day 5 and returned to the normal level at day 12. However, no humanSULT2B1b expression was detected in the liver of control group. Effectof SULT2B1b on PCNA expression in mouse liver tissues. PCNA is a proteinfor DNA replication in proliferating cells. To determine the effect ofSULT2B1b on proliferation in liver, PCNA was used as a proliferationmarker. Immunohistochemistry analysis showed that the number ofPCNA-positive cells in mouse liver tissues was significantly increasedwith the enhanced SULT2B1b expression, as compared to day 0 (FIGS. 20Aand B). However, in the control group, number of PCNA-positive cellsfollowing the infection did not change significantly (FIG. 20A). Westernanalysis in the FIG. 20C showed that the PCNA protein levels werecoincidently increased with SULT2B1b: started to increase at day 2,reached the maximum at day 5, and returned to the baseline at day 12.The results indicated that the increases in SULT2B1b expression inducePCNA expression, which further confirmed the immunohistochemistry study.

Locations of PCNA and SULT2B1b Expression in Mouse Liver Tissues.

To elucidate the relationship between SULT2B1b and PCNA, doubleimmunofluorescence was performed in liver specimens collected fromcontrol and SULT2B1b group at day 4 after infection. The results (notshown) indicated that the hepatocytes with PCNA expression in the nucleiwere accompanied by SULT2B1b overexpression in the cytosol.Interestingly, no PCNA expression was detected in the cells withoutSULT2B1b expression, indicating that SULT2B1b is directly associatedwith the expression of PCNA. The PCNA and human SULT2B1b positive cellswere undetectable in the liver sections from control group (data notshown).

Effect of SULT2B1b on the mRNA Levels of Cell Cycle Regulatory Genes inMouse Liver Tissues.

The mRNA levels of other proliferative genes were determined by RTqPCRin mouse liver tissues. As depicted in FIG. 21, when the expression ofSULT2B1b reached its highest level (day 4 or 5), the tissues producedstrong 2- to 4-fold increases in mRNA levels of PCNA (FIG. 21A),forkhead Box m1b (FoxM1b, (FIG. 21B), cell division cycle 25b (CDC25b,(FIG. 21C), and cyclin A (FIG. 21D). FoxM1b and its target gene CDC25bare known to be essential in cell cycle progression through G1/S andG2/M and cyclin A is a protein required for the cell to progress throughthe S phase. In addition, 2.6-fold increase of mRNA level for matrixmetalloproteinase-9 (MMP-9, FIG. 21E) was observed in mouse livertissues at day 5 after Ad-SULT2B1b injection. MMP-9 is a zincendopeptidase involved in the breakdown of extracellular matrix innormal physiological processes and tissue remodeling in the regeneratingliver. Thus, increases in SULT2B1b expression may promote liverproliferation in vivo.

Effect of SULT2B1b on LXR Activity and its Target Gene Expression in theMouse Liver Tissues.

To understand the possible mechanisms by which SULT2B1b promotes hepaticproliferation, the expression of genes regulated by LXR signalingpathway were analyzed in mouse liver tissues. As shown in FIGS. 22A andB, infection of mice for 2 to 5 d with Ad-SULT2B1b inhibited theactivity of LXR response in liver, the protein levels of LXRa and itstarget genes ABCA1 and SREBP-1c in the SULT2B1b group decreasedby >40%, >50%, and >70%, respectively. These results in vivo wereconsistent with previously reported studies showing inactive effect ofSULT2B1b on LXR response. Recent reports indicate that LXR activationinduces growth arrest and inhibits proliferation in many cells andanimal models. Thus, the stimulation of proliferation by SULT2B1b ismost likely via the inactivation of LXR signaling.

Effect of LXR Agonists on SULT2B1b-Induced Proliferation in Mouse LiverTissues.

Synthetic T0901317 and natural 25HC are LXR agonists. T0901317 canimpair SULT2B1b overexpression-induced LXR inactivation, while 25HCcannot. To determine whether the SULT2B1b-induced proliferation dependson LXR signaling repression, we compared the effect of these two LXRagonists in mouse liver. Briefly, mice with virus administration for 5days were given intraperitoneal injections of 25HC or T0901317 asdescribed in Methods. The results demonstrated that SULT2B 1 boverexpression indeed resulted in increased PCNA expression andrepressed LXR activation in the presence of vehicle or 25HC, as comparedto the corresponding control groups. However, the presence of T0901317significantly blocked the SULT2B1b-stimulated PCNA expression by 2- to4-fold (FIGS. 23A and B). These in vivo data clearly indicate theinvolvement of LXR signaling in SULT2B1b-induced proliferation.

Effect of SULT2B1b on Proliferation in Primary Rat Hepatocytes.

To study the effect of SULT2B1b on the hepatic proliferation geneexpression, the loss-of-function studies were performed usingsiRNA-SULT2B 1 b in PRH, which had shown relative high level of SULT2B1bexpression. As reported previously, rat SULT2B1b mRNA level in thecontrol group gradually increased with time in culture, and reached upto 35-fold at the 48 h (FIG. 24A). The expressions of cell cycleregulatory genes PCNA, FoxM b, CDK2, and cyclin A in the control groupwere also upregulated with time in culture (FIG. 24 B-E). However, whenthe SULT2B1b levels were efficiently (about 50%-70%) silenced followingsiRNA-SULT2B1b transfection (FIG. 24A), the mRNA levels of theseproliferative genes were substantially repressed (FIG. 24C-E). Theseresults demonstrated that PRH are potential proliferation cells andtheir proliferation capacity may be regulated by SULT2B1b expression. Asexpected, cell survival assay demonstrated an effective reduction ofcell viability in SULT2B1b-silenced hepatocytes, as compared to thecells infected with siRNA-Control and cultured in normal (N) (FIG. 24F), supporting our RTqPCR analysis. Effect of SULT2B1b on proliferationof PRH was also assessed by SULT2B1b overexpression. As shown in FIG.25A, infection of PRH with Ad-SULT2B1b at a MOI of 10 pfu/cell for 24h-48 h produced high SULT2B1b protein levels. Simultaneously, thehepatic proliferation potential was substantially promoted at 48 h asinvestigated through cell survival assay (FIG. 25B). RTqPCR showed thatin contrast with the loss-of-function results described above, SULT2B1boverexpression in PRH led to a further increase (about 2-fold) in themRNA levels of the proliferative genes PCNA, FoxM1b, CDK2 and cyclin A(FIG. 25C). Thus, similar to that in mouse liver tissues, SULT2B1bexpression may promote hepatocyte proliferation in vitro.

Effect of LXR Agonists on SULT2B1b-Induced Proliferation in PRH.

The role of LXR signaling in SULT2B1b-induced proliferation was furtherdemonstrated by in vitro application of T0901317 and 25HC in PRH. First,the effect of SULT2B1b on LXR signaling and PCNA protein levels wasinvestigated. As shown in FIGS. 26A and B, the LXR and SREBP1 proteinlevels in the control group gradually decreased with time in culture,and SULT2B1b overexpression further decreased the levels at 24 and 48 h.For the proliferative marker PCNA, Ad-SULT2B1b infection increased itsprotein level by 8-fold at 48 h (FIGS. 26A and B). Hence, 48 h waschosen for the following studies. As the western blot analysis shown inFIG. 29C, for the Ad-Control-infected group, treatment of cells for 48 hwith either T0901317 or 25HC significantly increased LXR response geneexpressions and decreased PCNA expression. Compared with cells treatedwith only virus (Ad-Control or Ad-SULT2B1b) infection for 48 h (FIGS.26A and B), the addition of 25HC results in a similar downregulation ofLXR response and upregulation of PCNA expression after SULT2B1boverexpression, while slight but no significant change was detected inthe presence of T0901317 (FIGS. 26 C and D), indicating that theactivation of LXR by T0901317 blocks the SULT2B1b-induced proliferation.These in vitro data further confirm that the effect of SULT2B1b onproliferation in vitro is LXR signaling pathway dependent.

DISCUSSION

In this study, we found that FoxM1b, CDK2, and cyclin A gene expressionsare significantly (>2-fold) increased in cultured PRH at 24-48 hcompared to 0 h. This increase in proliferative genes may play a role inhepatic survival in response to liver injury. Interestingly, SULT2B1bexpression is also enhanced (20-35 fold) with time in PRH culture, andSULT2B1b inhibition by siRNA results in significant downregulation ofthese cell cycle regulatory genes. The present data signify the factthat the increasing level of SULT2B 1 b expression is crucial for theproliferation of hepatocytes in culture.

We also showed that LXR signaling pathway is downregulated with time inPRH culture as seen by western blot. In fact, this downregulation of LXRresponse in PRH is similar to that in liver after partial hepatectomy:during liver regeneration, significant inactivation of LXR signaling isobserved following partial hepatectomy, and this is accompanied byincreases in SULT2B1b. In view of our results in normal mouse livers andprimary hepatocytes that SULT2B1b overexpression substantially repressesLXR response, it is reasonable to conclude that LXR signaling pathwaymay play a role in SULT2B1b-induced proliferation. Furthermore, the datain cultured hepatocytes showed that both LXR signaling activity and PCNAexpression are regulated by SULT2B1b, whereas the times differ: LXRinactivation occurs earlier than the upregulation of PCNA. This can beexplained based on our data where we studied the effect of LXR agonistT0901317 on SULT2B 1 b-induced proliferation in PRH. Considering thatthe enhanced PCNA protein level and repressed LXR response induced bySULT2B1b overexpression are blocked by T0901317, LXR signaling pathwaymust play an important role in SULT2B1b-induced proliferation in vitro.Thus, that the repression of LXR response occurs earlier (24 h) than theupregulation of PCNA expression (48 h) is not surprising.

We saw that SULT2B1b overexpression increased PCNA-positive cells andPCNA protein levels in a dose- and time-dependent manner in mouse livertissues. When LXR was activated in vivo, SULT2B1b retained part of itsability to induce PCNA. In fact, we also observed a direct relationshipbetween SULT2B1b and PCNA expression in vivo by doubleimmunofluoresence.

In summary, we for the first time provide convincing evidence thatSULT2B1b may promote hepatic proliferation via increasing cell cycleregulatory gene expressions in vivo and in vitro. By application of thesynthetic LXR agonist, we further found that LXR signaling is a keyfactor in SULT2B1b-induced proliferation. The results demonstrate apreviously undefined function of SULT2B1b in liver, and suggest a rolein regulating liver regeneration and the re-growth process.

Example 5. SULT2B1b Overexpression Promotes Liver Regeneration ViaInhibiting LXR Signaling in Primary Rat Hepatocyte and Mouse Liver withor without Partial Hepatectomy

In the present Example, we demonstrate that SULT2B1b plays a criticalrole in liver regeneration by inhibiting the LXR signaling pathway.Increases in SULT2B1b in mouse liver tissues with or without PHupregulate PCNA-positive cells in a dose- and time-dependent mannerwhile decreases in SULT2B1b significantly inhibit many proliferativegene expressions in PRH. Activation of LXR pathways in PRH by syntheticagonist completely blocks the SULT2B1b-induced increases in PCNA. Theresults provide novel therapeutic indications for liver injury,transplantation and surgery.

Materials and Methods Animals and Treatment.

C57BL/6 female mice were obtained from Charles River Laboratories(Cambridge, Mass.). Mice were hosted under a standard 12/12-hourdark-light cycle with standard rodent chow diet and water ad libitum.Nine- or 10-week-old mice were infected with recombinant adenovirusencoding CMV-driven SULT2B1b (lx 10⁸ pfu/mouse) through tail veininjection. Ad-CMV-Gal adenovirus was used as control. For the PH animalmodel, PH was performed under anesthesia following infection of the miceat the days as indicated in the text using Higgins and Anderson'smethods. Briefly, left lateral and median lobes were completely removed.For the sham-operated controls, an excision was made into the peritonealcavity followed by closure of the incision. At indicated times aftersurgery, mice were sacrificed by isoflurane inhalation, and serum andliver tissues were collected. To measure the liver re-growth rate, thecollected liver tissue from each group of mice (n=3-4) were weighed andcompared to the body weight at the time of sacrifice. To evaluate liverinjury, the liver-specific cytosolic enzyme activities of aspartateaminotransferase (AST), alanine aminotransferase (ALT) and alkalinephosphatase (ALP) in serum were measured using standard clinicalbiochemistry laboratory blood assays. All protocols were approved by theInstitutional Animal Care and Use Committee (IACUC) of the McGuire VAmedical center.

Culture of PRHPRH cultures, prepared as previously described (Hylemon etal., J Biol Chem 1992: 267; 16866-16871), were plated on 60-mm tissueculture dishes in Williams' E media containing thyroxine (1 μM) anddexamethasone (0.1 μM). Cells were infected with Ad-CMV-SULT2B1b at amultiplicity (MOI) of 10 plaque forming units/cell (pfu/cell) or 100 nMof siRNA-SULT2B1b (ON-TARGET plus siRNA of rat SULT2B1b, ThermoScientific Dharmacon, USA). Ad-CMV-13-Gal adenovirus and ON-TARGET piusnegative control siRNA were used as controls respectively.

Immunohistochemistry.

Liver tissues were fixed in 10% buffered formalin and embedded inparaffin. Deparaffinized 4-μm sections were stained with mouse anti-PCNAantibody (ab29, Abcam, MA, USA). Immobilized antibodies were detected bythe avidin-biotin-peroxidase technique (Vectastain ABC Kits, VectorLaboratories, UK). DAB was used as the chromogen and haematoxylin as thenuclear counterstain. The primary antibody was omitted as negativecontrol. For quantitation of PCNA expression, PCNA-positive andPCNA-negative nuclei were counted in at least five high-power fieldseach slide and expressed as the percentage of PCNA-positive cells.

Double Immunofluorescence.

Liver tissue in mice 4 days after Ad-SULT2B1b infection was used fordouble immunofluorescence to co-locate the expression of PCNA withSULT2B1b. Deparaffinized 4-μm sections were cultured with a cocktail mixof two primary antibodies: mouse anti-PCNA antibody (1:800; ab29, Abeam,MA, USA) and rabbit anti-SULT2B1b antibody (1:30; AB38412, USA).Subsequent antibody detection was carried out with secondary antibodies:Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen, USA) and AlexaFluor 488 goat anti-rabbit IgG (1:500; Invitrogen, USA). Negativecontrol was performed by replacing the primary antibody with PBS.Sections were examined with a fluorescence microscope and merged imageswere formed using Adobe Photoshop CS2.

Determination of Gene Expressions Involved in Proliferation and LXRPathway.

Total cell lysate was prepared from frozen mouse liver tissue or PRH,and 50 pg total proteins were subjected to 10% SDS-PAGE, transferred topolyvinylidene difluoride membranes (Millipore, Eschbom, Germany). Themembranes were probed with antibodies against PCNA, SULT2B1b (SantaCruz, Calif.), LXRa (Abcam, Cambridge, Mass.), ABCA1 (Abcam, Cambridge,Mass.), SREBP1 (Santa Cruz, Calif.) and actin (Sigma, USA) as a loadingcontrol for 2 h at room temperature. Western blot analysis was performedas previously described (Ren et al., J. Lipid Res. 2004; 45:2123-2131).

Total RNA was extracted using SV Total RNA Isolation Kit (Promega,Wisconsin, WI) according to the supplier's instruction. The relativemRNA levels were measured by RT-PCR and quantitative real-time PCR(RTqPCR) as previously described (Ren et al., J. Lipid Res. 2004;45:2123-2131). Expression levels were normalized to the 18S gene orGAPDH. Suitable oligonucleotide primers for RTqPCR were employed.

HPLC Analysis of Oxysterols in Liver Tissue.

Mouse liver samples (300 mg) that come from three biologically-distinctspecimens were pooled together to reduce variance and digested by 2mg/ml of Proteinase K in PBS (1 ml) at 50° C. for 12 h. 20 ml ofchloroform:methanol (2:1, v/v) was added to the digests, sonicated for30 min, and filtered, 4 ml of water was added, mixed, and allowed tostand for about 3 h for phase separation. The chloroform (bottom) phase,which mainly contains oxysterols, was added 30 μl of testosterone inchloroform solution (50 μg/ml) and evaporated under nitrogen stream. Theresidue was re-suspended in 8 mL of hexane and passed through apre-conditioned Waters Sep-Pak silica cartridge (400 mg) that had beenwashed with hexane (5 ml) and 1% isopropanol in hexane (5 ml). Thepurified oxysterols fraction was eluted with 8 ml of isopropanol:hexane(1:9, v/v) and evaporated under N₂.

The oxysterol samples thus obtained from the chloroform were oxidizedwith cholesterol oxidase. To the oxysterol sample dissolved in 50 μl of2-propanol were added 450 μl of water and 50 μl of 1M potassiumphosphate buffer (Kpi), and the resulting mixture was sonicated for 10min. To the mixture 4 units of cholesterol oxidase in Kpi buffer (10 μl)was added and incubated at 37° C. for 1 h. Methanol (300 μl) was addedto stop the reaction and the products, enones, were extracted with 3times with 2 ml of hexane, and the extracts were evaporated under an N₂stream. The residue was re-dissolved in 5% isopropanol in hexane (150μl) and 100 μl of the solution was subjected to the HPLC as describedbelow. HPLC analysis was conducted with Alliance 2695 separation modulefitted with 2487 Dual X absorbance detector (Waters, Milford, Mass.).The separation was carried out on an Ultraspere silica column (5 μm, 4.6mm id×250 mm; Beckman, Urbana, Ill.) and hexane:isopropanol:aceticacid=965:25:10 (by volume) as an eluent at a flow rate of 1.3 ml/min.The column temperature was kept constant at 30° C. and the enones weremonitored at the absorption at 240 nm.

Statistical Analysis.

Data are expressed as mean±standard deviation (SD). Statisticalcomparison was made using Student's t test for unpaired samples. A valueof P<0.05 was defined as statistical significance.

Results Effect of SULT2B1b on PCNA Expression in Mouse Liver.

Proliferation cell nuclear antigen (PCNA) is a critical protein requiredfor DNA replication in proliferating cells. To elucidate the growthregulation by SULT2B1b in liver, we used PCNA as the proliferationmarker. Mice were infected with Ad-SULT2B1b or Ad-control (1×10⁸ pfu)through tail vein injection. Mouse liver was harvested at 0, 2, 4, 5 and12 days after SULT2B1b infection. RTqPCR analysis showed that the mRNAlevel of SULT2B1b gene expression increased to its highest level (about150-fold) at the fourth or fifth day (D4 or D5) and returned steadily tothe normal level at the twelfth day (D12) (FIG. 27A).Immunohistochemistry analysis showed the number of PCNA-positive cellswas significantly increased following increases of SULT2B1b expressionin liver, as compared to DO (FIGS. 27B and C). Consistently, SULT2B1boverexpression also significantly increased the total protein levels ofPCNA. (FIG. 27D).

Co-Localization of PCNA and SULT2B1b Expression in Mouse LiverHepatocytes.

To elucidate the role of SULT2B1b in the expression of PCNA, mice wereinfected with Ad-SULT2B1b or Ad-control as described above. Four daysafter infection, PCNA and SULT2B1b expressions in paraffin sections ofmouse liver tissues were analyzed by double immunofluorescent staining(not shown). The merged images indicated that the hepatocytes with PCNAexpression in the nuclei are almost always accompanied by SULT2B1boverexpression in the cytosol, while there was no PCNA expression in thecells without SULT2B1b overexpression, indicating the directrelationship between SULT2B1b and PCNA. The number of PCNA and SULT2B1bpositive cell was difficult to detect in mouse liver infected withcontrol virus (data not shown).

Effect of SULT2B1b on Gene Expression Involved in Proliferation in MouseLiver.

To confirm the impact of SULT2B1b on promoting liver proliferation inmouse liver, the mice were infected with Ad-SIILT2B1b or Ad-control asdescribed above. RNA was exacted from liver tissue and the relative mRNAlevel of each gene was measured by qRTPCR. As the expression of SULT2B1breached to its highest level (at day 4 and day 5), the mRNA level offour well-known proliferative genes were substantially increased: PCNA,for DNA replication in proliferation cells; forkhead Box m1b (FoxM1b)and its target gene CDC25b, for the cell progression through GuS andG2/M phases; CyclinA, for GuS transition, and matrix metalloproteinase-9(MMP-9), a zinc endopeptidase, for hepatic matrix remodeling in theregenerating liver, and C myc (FIG. 28A-F).

Knockdown of SULT2B1b in PUB: Effect on Gene Expression for HepatocyteProliferation

To investigate whether SULT2B1b is crucial for hepatocyte proliferation,loss-of-function studies were performed using siRNA-SULT2B1b. Since ratliver expresses much higher SULT2B1b, PRH were selected for this study.During the culture of PRH, as expected, the levels of SULT2B1b graduallyincreased up to 35-fold at the 48 h time-point (FIG. 29A). Meanwhile,the expression of cell cycle related genes was also up-regulated (FIG.29B-E). After siRNA-SULT2B1b recombinant plasmid infection, SULT2B1bgene expression was successfully knocked down by 50-60% (FIG. 29A), andthe mRNA levels of proliferative genes CDK2, FoxM1b, CyclinA and PCNAwere significantly decreased (FIG. 29B-E).

Effect of PH on Expression of SULT2B1b and LXR Signaling Pathway.

PH is the best model for investigating liver regeneration. In order tounderstand the mechanism of SULT2B1b in promoting proliferation, we useda mouse PH model to detect the expression of SULT2B1b and genes involvedin the LXR signaling pathway. Mouse liver tissues were harvested at 0,1, 3 and 5 days after PH. Total RNA was exacted from liver tissue andgene expression (as mRNA levels) was measured by PCR and RTqPCR.Consistent with previous findings, once PH is performed on a mouse, theliver begins to regenerate, as evaluated by the mRNA level of PCNA (notshown). Interestingly, SULT2B1b expression was significantlyup-regulated during this process (not shown), and was accompanied by thesuppression of LXR response gene expression, including ABCA1, ABCG1 andSREBP1, as compared by sham-operated mice (not shown). It is possiblethat the promotional effect of liver proliferation by SULT2B1b is viainhibiting LXR signaling pathway.

Effect of SULT2B1b on Hepatocyte Proliferation Via LXR Signaling Pathway

To determine that the effect of SULT2B1b on liver proliferation isthrough the LXR signaling pathway, we compared the gene expressionsinvolved in LXR signaling pathway between the mice infected withAd-SULT2B1b and Ad-control mice. As expected, overexpression of SULT2B1bsignificantly decreased protein levels of LXRa, ABCA1 and SREBP-1 inmouse liver (FIG. 30A). RTqPCR analysis also showed that SULT2B1boverexpression significantly decreased mRNA levels of LXR target genesSREBP-1, ABCA1, ABCG1 and ABCG5 in mouse liver tissues (FIG. 30B-E). Theresults were consistent with those in PRH (not shown). In the cells withSULT2B1b overexpression, the protein level of PCNA was significantlyincreased after 48 hours in culture, while the protein levels of LXRcL,ABCA1 and SREBP-1 were significantly decreased at both of 24 and 48 hourtime-points (FIG. 31A). SULT2B1b overexpression also increased mRNAlevels of PCNA, FoxM1b, and CDK2 (FIG. 31B-D), and decreased mRNA levelsof SREBP1, ABCG5 and ABCA1 compared with those in the cells infectedwith Ad-ct virus (FIG. 6F-11).

Previous studies showed that, T090 1317, as a synthetic agonist of LXR,effectively blocked the effect of SULT2B1b on LXR in human aorticendothelial cells, but not 25HC, the natural ligand which can besulfated to 25HC3S by SULT2B1b (Bai et al. Atherosclerosis 2011;214:350-356). To confirm that the impact of SULT2B1b on proliferation isLXR dependent, T0901317 and 25HC were used to compare the effect in PRH.As previously reported, in the cells with control virus infection, bothagonists increased LXR response gene expressions, however, the effectsof 25HC were impaired by overexpression of SULT2B1b (FIG. 31A).Interestingly, the presence of 25HC further increased the expression ofPCNA stimulated by SULT2B1b overexpression, but T0901317 blocked thestimulation by SULT2B 1 b (not shown).

Effect of SULT2B1b Overexpression on Hepatocyte Proliferation DuringLiver Regeneration

To investigate whether SULT2B1b overexpression stimulates hepatocyteproliferation during PH-induced liver regeneration, we infected PH micewith Ad-SULT2B1b or Ad-Control (lx 10⁸ pfu). Livers were harvested andweighed for calculating liver regrowth. Interestingly, mice infectedwith Ad-SULT2B 1 b exhibited a faster liver regrowth during the first 3days after PH and taking only 3 days to achieve their original masswhile control mice took 5 days to do so (FIG. 32A).

To better understand if SULT2B1b overexpression exerts an impact on thehepatocyte proliferation response, PCNA immunostaining was performed inliver sections. At each time point after PH, the number of PCNA-positivehepatocytes in Ad-SULT2B 1 b infected mice was substantially increasedcompared with Ad-Control infected mice (FIG. 32C). Consistently, micewith SULT2B1b overexpression showed significant increases in the proteinlevel of PCNA expression, compared to controls (FIG. 32B). To evaluatewhether acute liver injury might influence the regeneration, serum AST,ALT and ALP levels were measured. Compared with Ad-Control infected micegroup, strong decreases of AST, ALT and ALP serum levels in AdSULT2B1binfected mice were observed 1 or 3 days after PH (FIG. 32D-F),indicating SULT2B1b significantly decreased liver damage by suppressinginflammation responses. Thus, SUTL2B1b acts positively on liverproliferation after PH, both promoting the hepatocyte proliferation rateand exerting a specific reduction of acute liver injury.

Effect of SULT2B1b Overexpression on Hepatocyte Proliferative GeneExpression During Liver Regeneration.

The promotion in hepatocyte proliferative capability by Ad-SULT2B1binfection during liver regeneration was confirmed by RTqPCR. SULT2B1boverexpression led to a significant increase in the expression ofproliferative genes involved in the regulation of cell cycleprogression, PCNA, CyclinA, FoxM1b, C-myc and CDC25b (FIG. 33A-E).Significant increases in the expression of extracellular matrixremodeling gene MMP-9 was also detected in AdSULT2B1b infected mice, ascompared with Ad-Control mice (FIG. 33G). These data confirm that theoverexpression of SULT2B1b in the regenerating liver promotes hepatocyteproliferative capability.

Reduction of Hepatic Oxysterol Levels by SULT2B1b Overexpression afterPH

In order to investigate whether the down-regulation of the LXRtranscriptome is associated with reduction of endogenous oxysterolligands, we measured the hepatic oxysterol levels at day 0 and 3 afterPH. In the Ad-SULT2B1b infected mice, the hepatic amounts of7-ketocholesterol, 25-hydroxycholesterol, and 6β-hydroxycholesterol wereapparently lower compared to those of Ad-Control mice (Table 7). Thisdata reveals a scenario wherein the presence of SULT2B1b there is anapparent reduction of oxysterols coupled with a down-regulation of theL×R system, thus contributing to the significant improvement inproliferative capability in regenerating liver.

TABLE 7 Oxysterols in liver tissue Oxysterol 0 days after PH 3 daysafter PH (ng/g liver) Control SULT Control SULT 7KC 1060.1 460.8 1190.5581.2 25-HC 249.5 214.7 228.6 165.3 6β-HC 1183.8 447.4 940.3 537.9 27-HC49.04 — — —

DISCUSSION

In this report, using PRH and mouse liver with or without PH, we for thefirst time show that SULT2B1b can promote hepatocyte proliferation.Overexpression of SULT2B1b leads to dramatic increases in the number ofPCNA-positive cells in mouse liver with or without PH.Double-immunofluscence provides strong evidence that the increase inPCNA expression is directly related to SULT2B1b overexpression.Furthermore, SULT2B1b overexpression in PP. 11 and in mouse liver withor without PH significantly increases expression for a variety of liverproliferative genes PCNA, Cyclin A, FoxM1b, CDC25b, C-myc and CDK2,which are important markers and regulators in proliferation and cellcycle. Conversely, knockdown of SULT2B1b inhibits proliferation in PRH.These results suggest that SULT2B1b is a critical factor in regulatingliver proliferation.

These studies also indicate that the effect of SULT2B1b on liverproliferation is also highly associated with the suppression ofLXR-driven pathway, as manifested by the decreased expression of LXR andits target genes, SREBP1, ABCA1 in the regenerating liver and throughoutPRH culture, in both of which can be detected significant increases inthe proliferative genes, such as PCNA, CyclinA, CDK2 and FoxM1b.

We detected PCNA expression after SULT2B1b overexpression in PRH in thepresence of 25HC or T0901317 and confirmed that overexpression ofSULT2B1b in cultured cells impairs LXR response to 25HC but does notalter the receptor response to T090 1317. Simultaneously, PCNAexpression is significantly increased in the presence of 25HC, while nosignificant change of PCNA expression is observed in the presence ofT0901317 following overexpression of SULT2B1b. These data indicate thatthe suppression in LXR response provides a critical step for the actionof SULT2B1b on hepatocyte proliferation. SULT2B1b appears to inactivateoxysterols as LXR ligands, thereby inhibiting LXRs signaling pathway,and subsequently activating the expression of cyclin-dependent kinases,promoting cell proliferation.

In this study, SULT2B1b overexpression significantly increased the keycell cycle related genes, CyclinA, CDK2 and FoxM1b in both RPH and mouseliver with or without PH. Furthermore, by comparing the serumcholesterol levels in the mouse with Ad-SULT2B1b or Ad-control infection(Data not shown), we confirmed that the promotional effect of SULT2B1bon liver proliferation is independent of cellular cholesterol levels,because a significant decrease in serum cholesterol levels is detectedin the SULT2B1b overexpression mouse group.

In summary, this study provides strong evidence for the first time thatSULT2B1b promotes hepatocyte proliferation and liver regeneration viainhibiting LXR signaling pathway in PRH and mouse liver with or withoutPH. These findings suggest that SULT2B1b plays a crucial role in liverregeneration and suggest novel therapeutic measures for livertransplantation and surgery.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-25. (canceled)
 26. A method of treating a subject with hepatitiscaused by consumption of alcohol comprising administering to the subjecta composition comprising a therapeutically effective amount of25-hydroxycholesterol-3-sulfate (25HC3S) and a pharmaceuticallyacceptable excipient.
 27. The method of claim 26, wherein the 25HC3Scomprises a sodium salt thereof.
 28. The method of claim 26, wherein thepharmaceutically acceptable excipient comprises water.
 29. The method ofclaim 26, wherein the composition further comprises a pH bufferingagent.
 30. The method of claim 26, wherein the administration comprisesat least one of oral administration, enteric administration, sublingualadministration, transdermal administration, intravenous administration,peritoneal administration, parenteral administration, administration byinjection, subcutaneous injection, and intramuscular injection.
 31. Themethod of claim 26, wherein the administration comprises at least one oforal administration and intravenous administration.
 32. The method ofclaim 26, wherein the 25HC3S is administered in an amount ranging from0.1 mg/kg to 100 mg/kg, based on body mass of the subject.
 33. Themethod of claim 26, wherein the 25HC3S is administered in an amountranging from 1 mg/kg to 10 mg/kg, based on body mass of the subject. 34.The method of claim 28, wherein the 25HC3S comprises a sodium saltthereof.
 35. The method of claim 29, wherein the 25HC3S comprises asodium salt thereof.
 36. The method of claim 30, wherein the 25HC3Scomprises a sodium salt thereof.
 37. The method of claim 31, wherein the25HC3S comprises a sodium salt thereof.
 38. The method of claim 32,wherein the 25HC3S comprises a sodium salt thereof.
 39. The method ofclaim 33, wherein the 25HC3S comprises a sodium salt thereof.
 40. Themethod of claim 28, wherein the administration comprises at least one oforal administration and intravenous administration.
 41. The method ofclaim 29, wherein the administration comprises at least one of oraladministration and intravenous administration.
 42. The method of claim32, wherein the administration comprises at least one of oraladministration and intravenous administration.
 43. The method of claim33, wherein the administration comprises at least one of oraladministration and intravenous administration.